Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDE DERIV~TIVES
The present invention relates to certain novel peptide derivatives which possessph~rrnz3cologically useful plop..lies for use in treating auLoi~ une ~iiee~ees or medical
conditions, such as rh~m~toid art_ritis and other MHC class II dependent T-cell mediated
~liee~eçe The invention also includes ph~rm~ceutical compositions of the novel chemical
GompouAAnAds~ proce~ses for tAhAeiAA m~n~ t~- e, ~nd tllAei; use in tLeating ûne or r~AOre of ire
aforementioned diseases or medical conditions and in the production of novel pharm~- ellticals
for use in such medical tre~tmPnte
Stimulation of the human immune response is dependent on the recognition of protein
antigens by T cells. However T cells cannot respond to antigen alone and are only triggered
by antigen when it is complexed with major histocompatibility complex (MHC~) molecules on
the surface of an antigen ~l~sP~ 1g cell, such as a B cell, macrophage or dendritic cell.
MHC class I molecules elicit a T-killer cell response which results in the destruction
of the cell bearing the antigen. MHC class II molecules elicit a T-helper cell response which
controls the expansion and maturation of selected B cells (i.e. generation of antigen-specific
antibodies) and activation of macrophages.
A critical requirement of the immllne system is the ability to differentiate between
"self' and ~'non-self' (i.e. foreign) antigens This tiieerimin~tion is required to enable the
immune system to mount a response to invading foreign pathogens whilst m~int~ining
tolerance to self-proteins and thereby pl~ ing darnage to normal tissues. An autoimml-n~
disease results when self-tolerance breaks down allowing the immune system to react against
sel~-tissues such as the joints in rh~ m~3toid arthritis. It is thought that the m~int~n~nc:e of
tolerance and thus avoidance of autoimmllne disease is critically dependent on the function of
MHC molecules.
The observation that many auloi-"~ iee~e~s are linked to the inheritance of
particular MHC alleles suggests a key role for MHC molecules in the pathogenesis of
uLoi ~ . ", 1llnc disease. For instance multiple sclerosis is linked to the inheritance of HLA-DR2,
insulin dependent diabetes mellitus to HLA-DR3 and/or HLA-DR4 and Hashimoto's
thyroiditis to HLA-DR5. In particular, an especially strong association exists between
predisposition to development of the chronic infl~mm~tory joint disease rheumatoid arthritis
and the inheritance of HLA-DR4Dw4 and/or HLA-DR4w14 and/or HLA-DR1. It is thought
that the autoimmune disease associated MHC molecules bind to certain self-antigens and
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present them to T cells thus stim~ tin~ an J~ o;....~.~m~ response. Other peptides which can
bind to the ~uLoi~ associated MH~ molecules and/or either prevent the binding or
displace already bound self-antigens and/or which inhibit T cell activation (especially the
activity of pathogenic T cells (e.g. Th 1 cells)) and/or which increase the activity of protective
S T cells (e.g. Th 2 cells), or peptides which interact with MHC molecules by an alternative
mech~ m of action so as to prevent or modify stim~ tion of an autoimm~m~ response
mediated via said MHC molecules, may specifically suppress an autohll,ll-ule response.
An agent of this kind would offer therapy for the autoimmlme disease whilst avoiding
general ~pl~ssion ofthe immllne system, thus limitinp deleterious side-effects. This kind of
10 profile would have significant advantages over current therapy for fiice~eeC such as
rheurnatoid arthritis. It is contemporary practice to treat rheumatoid ar~hritis initially with
symptom relief agents such as NSAIDs, which do not have any beneficial effect on disease
progression and are often associated with unwanted side-effects. Tre~m~nt of more severe
disease relies on the use of the so-called second-line agents. Often these are general cytotoxic
15 compounds which are of limited efficacy and can cause severe toxicity problems. A rationally
based, disease modifying agent, without associated non-specific cytotoxicity, would therefore
offer significant ~enefits in the treatment of rh~llm~toid arthritis.
Peptides are disclosed in International Patent Application, Publication Nos. WO
g2/02543, WO 93/05011 and WO 95/07707 which bind MElC molecules and inhibit T-cell
20 activation.
Although a nurnber of peptides have been discovered which inhibit HLA-DR restricted
T cell activation by binding to HLA-DR molecules, there is a co.~ lling need for alternative
compounds which bind to such molecules and/or either prevent the binding or displace
already bound self antigens and/or inhibit T-cell activation and/or increase the activity of
25 protective T-cells, or which interact with MHC molecules by an alternative mechanism of
action, so as to prevent or modify stim~ tion of an auto~ c response that causes a
disease or condition referred to above.
We have discovered that the peptide derivatives of the present invention (set out
below) surprisingly possess such pharrnacologically useful properties and this is a basis for
30 the present invention.
According to one aspect of the invention there is provided a peptide aerivative of the
formula I (set out hereinafter)
-
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-3-
wherein
P is a hydrophobic residue; Rl is a sequence of 5 L-amino acids and R3 is a single L-amino
acid; or R' is a sequence of 3 L-amino acids and R3 is a sequence of 3 L-amino acids;
R2 is a group of the formula II (set out hereinafter) or III (set out hereinafter) in which Ra and
Rb are independently selected from hydrogen and ( 1 -4C)alkyl and A is methylene (CH2) or
oxygen; and
R4 is OH, NH2 or NRcRd wherein Rc is selected from (1-4C)alkyl, 2-carbamoylcyclopentyl,
2-pyridylmethyl, 4-carbamoylcyclohexyl, 4-carbamoylcyclohexylmethyl, 3-carbamoylphenyl,
4-carbarnoy~phenyl, 4-(carbamoylmethyl)phenyl, 4-(carboxymethyl)phenyl, 4-
(methoxycarbonylmethyl)phenyl, 2-morpholinoethyl and a group of the forrnula -Al-Gl in
which Al is (3-7C)alkylene or
A' is selected from
( 1 ) a group of the forrnula -A2-B2- in which A2 is p-phenylene or 1 .4-cyclohexylene and B2 is
(1-4C)alkylene or A2 is methylene and B2 is p-phenylene or 1,4-cyclohexylene; and
(2) a group of the formula -A3-B3-C3- in which A3 is methylene, B3 is p-phenylene or 1,4-
cyclohexylene and C3 is (1-3C)alkylene; and
Gl is a group of the forrnula -N=C[N(Rp)2}2 in which each Rp is independently selected from
hydrogen, methyl, ethyl and propyl;
or Al is a group of the formula -A4-B4- in which A4 is ~-phenylene and B4 is -CH2-CO- and
~l is 2-morpholinoethyl or 4-[2-(2-hydroxyethoxy)ethyl]~i~e.d;Gil1- 1 -yl:
and Rd is hydrogen or (1-4C)alkyl; or
R4 is 1-pipti,~illyl, 4-methyl-l-piperazinyl, 4-amidino-1-pil,eld,illyl, 4-(2-(2-
hydroxyethoxy)ethyl)- l -yi~ ~inyl, I -piperidyl or 4-substituted- 1 -piperidyl wherein the 4-
sllhstit~t~nl is selected from carboxy, carbamoyl, N-(2-aminoethyl)carbamoyl and N-(4-
aminobutyl)carbamoyl; or
R4 is a sequence of 1 to 6 amino acids or an atnide thereof; or a ph~rrn~eutically acceptable
salt thereof.
~ It is to be understood that an amino acid of R4 may independently have the D- or L-
stereochemistry. Furthermore, when R4 is defined as hydroxy (OH), this will be understood to
be the hydroxy group of the C-terminal amino acid of R3. Similarly where R4 is defined as
N~l2, NRcRd, piperazinyl, piperidyl; etc., this means that the hydroxy group of the C-tçrminz~l
amino acid of R3 is replaced by such a group. It is also to be understood that where an amino
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acid is referred to this means an alpha-amino acid. It is also to be understood that when an L-
arnino acid is referred to this also includes amino acids such as Gly, 2,2-diethylGly, aza-
alanine and a_a-glycine which have no chiral carbon atom. It is further to be understood that
generic terms such as "aL~cyl" include both straight and l~ chcd chain variants when the
5 carbon numbers permit. The same convention applies to other radicals.
It is well known in the art that compounds having a chiral centre may exist in the forrn
of a racemate (or a mixture of diastereoisomers where there is more than one chiral centre) or
as an optically active enantiomer or diastereoisomer. It is also well kno~,vn in the art that a
particular biological activity associated with a racemic or diastereomeric mixture may result
10 largely or solely from a single optically active isomer. It will therefore be understood that the
invention concerns any form of a peptide derivative of formula I ~hich possesses the
aforementioned ph~rrn~ceutically useful properties. It is well known in the art how to obtain a
single optically active isomer, for example by separation from a racemic or diastereomeric
mixture cont~ining the isomer using conventional techniques. such as chromatography, or by
15 chiral synthesis using an 2~lopl;ate optically active starting material or intermediate, as
exemplified herein. It is also well knov~n in the art how to deterrnine the ph~rrn~rological
,Lies of such racemic or diastereomeric mixtures, and the individual optically active
isomers, for example by using the assays described herein. The person skilled in the art is
therefore easily able to obtain the particular isomers of the peptide derivatives of formula I
2Q having the beneficial ph~rrn~r,ological pl~pc~lies referred to herein.
It is also to be lln-l~rctood that the present invention also encompasses any
polymorphic form, any tautomer or any solvate, or any mixture thereof. of a peptide derivative
-of formula I which l~o~esses the benefici~i ph~ cological prop~rties referred to herein.
Suitable independent values for the o~-arnino acids comprisin~ R' and R3 include, for
25 exarnple, the 20 naturally occl-rring amino acids encoded by the genetic code, particularly
alanine (Ala), glutarnic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), Iysine (Lys),
asparagine (Asn), glutamine (Gln), arginine (Arg), threonine (Thr). valine ~Val) and proline
(Pro). Amino acids such as sarcosine (Sar), 3,3,3-trifluoroalanine. 2.~-diethylglycine, 2,3-
min~propanoic acid (Dap), 2,4-diaminobutanoic acid (Dab), 2-arninobutanoic acid (Abu),
30 homoarginine, homophenylalanine, trans-4-hydroxyproline (Hyp) aza-alanine
LH2N-N(CH3~-COOH; A_ala], ~7~-glycine ~H2N-NH-COOH: Azgly]. 1.2.3,4-
- tetrahydroisoquinoline-3-carboxylic acid (Tic). octahydroindole-~-carboxylic acid ~Oic)~
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decahydroisoquinoline-3- carboxylic acid (Dic) are also suitable. (Where Dic is referred to
this means the forms in which the ring-junctions both have the R configuration or both have
the S configuration.) Gll.G:,lJol1ding N2-methylated amino acids may also be used, as well as
corresponding amino acids in which a free side-chain carboxylic acid function is esterified
5 (for example as an ( 1 -6C)alkyl or benzyl ester) and a free side-chain amino group is alkylated
~for example, methylated), acetylated or converted to a carbamate (for exarnple, an alkyl (such
as methyl or ethyl), phenyl or benzvl carbamate). Other suitable values for Rl and R3 include,
for example, 2-substituted glycine in which the 2-substituent is a group of the formula -
(CH2)sNH2 wherein s is 1 to 3, or a group of the formula -(CH2)pN(Re)3 .X~ wherein p is 2 to
10 4 and X is a counter ion (such as acetate, trifluoroacetate, hydroxide or chloride), or a group
of the formula -(CH2)qN(Re)2 wherein q is 0 to 4 or a group of the formula
-(CH2)rN=C[N(Re)212 wherein r is 1 to 4, wherein in which last three groups each Re is
independently selected from hydrogen and (1-4C)alkyl (such as methyl or ethyl).
A value for Rl of particular interest when it is a sequence of 5 amino acids includes,
15 for example, a sequence in which the fifth arnino acid (as read from left to right) is Val or Thr
and a seqllent~e in which the fourth and fif~Lh amino acids is Lys-Val, Arg-Val, Lys-Thr, Arg-
Thr, Ala-Val or Ala-Thr.
A particular value for Rl when it is a sequence of 5 amino acids includes, for example,
Ala-Ala-Ala-Lys-Val, Ala-Lys-Ala-Ala-Val, Ala-Ala-Ala-Arg-Val, Ala-Arg-Ala-Ala-Val,
20 Ala-Lys-Ala-Lys-Val, Ala-Arg-Ala-Arg-Val, Ala-Arg-Ala-Lys-Val. Ala-Lys-Ala-Arg-Vah
Ile-Ala-Ala-Arg-Thr, Arg-Ala-Ala-Ala-Val, Arg-Ala-Ala-Ala-Thr, Ala-Ala-Ala-Arg-Thr,
Ala-Arg-Ala-Arg-Thr, Ala-Ile-Ala-Arg-Val, Ala-Arg-Ala-His-Val, Ala-Arg-Ala-Ala-Thr,
Ala-Ala-Asn-Arg-Val or X-Ala-Ala-Ala-Thr where X is -NH.CH~CH2NH.C(=NH).NH2].CO-(he~Gil~n~,. referred to as "Gap"), or -NH.CH(CH~N=CtN(CH3)2]2).CO- ~hereinafter referred
25 to as "GapMe4"~, of which Ala-Ala-Ala-~ys-Val, Arg-Ala-Ala-Ala-Val, Arg-Ala-Ala-Ala-
Thr, Ala-Ala-Ala-Arg-Thr, Ala-Arg-Ala-Arg-Thr, Gap-Ala-Ala-Ala-Thr and GapMe4-Ala-
Ala-Ala-Thr are preferred. Arg-Ala-Ala-Ala-Val and Arg-Ala-Ala-Ala-Thr are particularly
~11 G rG I I ~Gd .
A value for R' of particular interest when it is a sequence of 3 amino acids includes,
30 for example, a sequence in which the amino acid adjacent to R2 is Ala and a sequence in
which the second and third amino acids (as read from left to right) is Lys-Ala, Arg-Ala, Ile- ~
Ala and Ala-Ala.
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A value for ~1 of special interest when it is a sequence of three amino acids includes,
for example, Ala-Lys-Ala, Ala-Arg-Ala, Arg-Ala-Ala, Arg-Ile-Ala and Ile-Arg-Ala,~speci~lly Ala-Arg-Ala.
A value for R3 of particular interest when it is a sequence of three amino acidsincludes, for example, a sequence in which the first amino acid (as read from left to right, i.e.
adjacent to R2)is Ala or Leu.
A plcf~L.ed value for R3 when it is a sequence of three amino acids includes, for
example, Ala-Ala-Ala, Leu-Arg-Ala and especially Ala-Arg-Ala.
~ pl~;~.led value for R3 when it is a single amino acid includes, for example, Ala, Gly
10 and Azgly, especially Ala .
Particular values for Ra and E~b when they are alkyl include, for example. methyl,
ethyl and propyl.
A p.ert;lled value for Ra and Rb includes, for example, hydrogen and methyl.
A suitable value for the hydrophobic residue P (which it will be appreciated is attached
15 to the amino group of the N-t~rmin~l amino acid of ~l) includes~ for example, an organic
hydrophobic group such as a hydrophobic aliphatic~ aromatic, heteroaromatic or mixed
aliphatic/aromatic or aliphatic/heteroaromatic organic group of from 5 to 20 carbon atoms
(and 1, 2 or 3 heteroatoms selected from oxygen, sulphur and nitrogen for heteroaryl-
c~mt~inin~ groups), for example a group of the formula R-, R.CO-. R.SO2-~ R.O.CO-.
20 R.NHCO-, R.O.CS-, R.S.CO-, R.NHCS-. R.S.CS- and R.CS-, in which R includes, for
eY~mple, (S-lOC)alkyl, aryl, heteroaryl, aryl(2-lOC)alkyl, heteroaryl(2-lOC)alkyl, diaryl(2-
8C)aL~cyl, aryl(2-1~C)alkenyl, arylcyclopropyl, (5-lOC)cycloalkyl, (5-lOC)cycloalkyl(2-
6C)aL~cyl, 3-biphenyl, 4-biphenyl, 4-cyclohexylphenyl, 2-naphthyloxymethyl, 3-
naphthyloxymethyl, phenoxyphenyl and tetrahydronaphthyl, an aryl or heteroaryl group of
25 which values of R may bear one or more ( 1 -4C3alkyl, halogeno, cyano or ( 1 -4C)alkoxy
sllbstitll~ntc One particular embodiment of the invention includes, for example. peptide
derivatives of the formula I in which P is R.CO- as defined above. A further particular
embodiment of the invention includes, for example, peptide derivatives of the formula I
wherein P is a hydrophobic aliphatic, aromatic or aliphatic/aromatic organic group of from 5
30 to 20 carbon atoms.
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Particular values for R include, for ~x~ .lc, when it is (S-lOC)alkyl: pentyl, isopentyl,
s-pentyl, 2-methylpentyl, hexyl, isohexyl, 5-methylhexyl and octyl; when it is aryl: phenyl,
naphthyl and indenyl; when it is heteroaryl: 2-, 3-, 5- or 6-indolyl, 2-, 3-, 5- or 6-indolinyl,
2-, 3-, 5- or 6- benzo[b]thiophenyl, thienyl, 2-, 4- or S-benzothiazolyl, 2-, 4- or 5-
S benzoxazolyl, 2-, 4- or S-benzimidazolyl, 1,4-benzodioxanyl ~qtt~ehe~l at the 2-, 3-, 6- or 7-
position and 2-, 3-, 5- or 6-benzofuranyl; when it is aryl(2-lOC)alkyl: aryl(2-6C)alkyl (where
the aryl portion includes, for example, any of the specific values for aryl given above and the
(2-6C)alkyl portion includes, for example, methylene, ethylene, trimethylene, tetramethylene
and pentarnethylene) such as 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl and 5-10 phenylpentyl; when it is h~Le~ ~yl(2-lOC)alkyl: hel~ 3~yl(2-6c)alkyl (where the heteroaryl
portion includes, for exatnple, any of the specific values for heteroaryl given above and the (2-
6C)alkyl portion includes, for exarnple~ methylene, ethylene, trimethylene, tetramethylene and
pentamethylene) such as in 2-~2-cyanobenzo~b]thiophen-5-yl)ethyl; when it is diaryl(2-
8C)alkyl: diaryl(2-6C)alkyl such as 2,2-diphenylethyl, 3,3-diphenylpropyl and 4,4-
15 diphenylbutyl; when it is aryl(2-lOC)alkenyl: aryl(2-6C)alkenyl such as styryl, 3-
phen~ v~ l-2-yl a~-ld 4-pheny;buten-i-yi, when it is aryicyciopropyi: phenylcyclopropyl, 1-
naphthylcyclopropyl and 2-naphthylcyclopropyl; when it is (S-lOC)cycloalkyl: cyclopentyl,
cyclohexyl and l-~ m~ntyl; and when it is (S-lOC)cycloalkyl(2-6C)alkyl:
2-(cyclohexyl)ethyl, 3-(cyclohexyl)propyl and 4-(cyclohexyl)butyl. A particular value for a
20 sllbstit~lent on an aryl group of R includes, for exarnple, methyl, ethyl, chloro, ~romo, iodo,
methoxy, ethoxy and cyano.
The hydrophobic residue P also includes, for example, a hydrophobic L-amino acid,
such as phenylalanine (Phe) and hydrogenated analogues thereof such as cyclohexylalanine
~Cha), para-chloroPhe, 3-(2-thienyl)~l~nin~, tyrosine (Tyr), Tyr(Omethyl), tryptophan (Trp),
biphenyl~l~nint~ 3-(1-naphthyl)~l~nin~, 3-(2-naphthyl)alanine and hydrogenated analogues
thereof, 3-( l-~ l)alanine (Ada), Glu(OBenzyl), 3-(benzyloxy)Ala, 3-
(benzylsulfanyl)Ala and 9-fluorenylGly, each of which may optionally bear on the N-termin~-~
a hydrophobic aliphatic, aromatic, het~ 3~onlatic or mixed aliphatic/aromatic oraliphatic/heteroaromatic organic group as defined or exemplified above. Alternatively, the
hydrophobic amino acid may optionally bear, for example, a further sequence of l to 3 amino
acids selected from any of the values for R' and R3 defined above. For example P includes
-
-
-
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the particular sequences ~la-~ha, Ala-Ala-Cha, Tyr-Ala-Ala-Cha, Tyr-Ala-Ala-Phe, Ala-Phe-
Phe-Phe and Ala-Ala-Ala-Phe. The first arnino acid of such further sequence of 1 to 3 arnino
acids (as read from left to right) may be an L- or D-arnino acid and may also optionally bear a
hydrophobic aliphatic, aromatic, heteroaromatic or mixed aliphatic/aromatic or
aliphatic/heteroaromatic organic group as defined or exemplified above.
Further particular values for P inchlclc, for example, 3-(benzyloxycarbonyl)propionyl-
Phe, 3-~benzyloxycarbonyl)propionyl-Cha, 4-(benzyloxycarbonyl~butyryl-Phe, 4-
(benzyloxycarbonyl~butyryl-Cha, (5-oxo-pyrrolidin-2-yl~carbonyl-Phe-Tyr,, (5-oxo-
pyrrolidin-2-yl)carbonyl-Glu(OBenzyl~-Tyr, acetyl-Glu~OBenzyl)-Tyr,
diphenylmethyl.CONH.CH2CH2.CO-Cha, diphenylmethyl.CONH.CH2CH2.CO-Tyr,
diphenylmethyl.CONH.CH2CH2CH~.CO-Cha, diphenylmethyl.CONH.CH2CH~CH7.CO-Tyr,
diphenylmethyl.NHCO.CH2CH2CH2.CO-Cha, diphenylmethyl.NHCO.CH2C~I2CH2.CO-Tyr,
benzyl.NHCO.CH2CH2.CO-Cha, benzyl.NHCO.CH2CH2.CO-Tyr. N-acetyl-4-chloro-beta-
hydroxyPhe, 4-phenoxyphenyl.NHCO-, benzyl.NHCO.CH2CH2.CO.(N-methylPhe),
benzyl.NHCO.CE~2CH2.CONH.CH(CHPh2).CO, benzyl.NHCO.CH2CH2.CO-Tyr,
3,3-diphcllyl~lu~ionyl, trans-cinnamoyl, 5-phenylvaleryl and 3-(2-cyanobenzo~b]thiophen-5-
yl)propionyl.
A value for P of particular interest includes, for example, Ph.(CH2)4.CO-
~5-phenylvaleryl (Phv)), Ph.(CH2)4.CS- and 3-(2-cyanobenzo[b]thiophen-5-yl)propionyl.
A preferred value for the hydrophobic residue P includes, for example, 3-(2-
cyanobenzo[b]thiophen-5-yl)propionyl and S-phenylvaleryl (Phv), especially the latter.
When Rc is a group of the forrnula -Al-GI, a particular value for Al when it is
alkylene includes, for example, methylene, ethylene, propylene and butylene; a particular
value for B2 when is is (1-4C)alkylene includes, for example, methylene, ethylene and
propylene; and a particular value for C3 when it is (1-3C)alkylene includes, for example,
methylene, ethylene and propylene.
A particular value for -Al-GI includes~ for example, 3-guanidinopropyl, 4-(2-
guanidinoethyl)phenyl, 4-(2-morpholinoethyl.NH.CO.CH2)phenyl and 4-(4-[2-(2-
hydroxyethoxy)ethyl]piperazin- I -yl.CO.CH2)phenyl.
A particular value for R4 when it is a sequence of I to 6 amino acids or an amide
thereof, includes, for example~ a sequence of L- amino acids independently selected from any
of the values for R' and R3 defined above (such as Ala-Thr-Gly-OH), or their D- analogues, or
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_ 9 _
a sequence conf~ining both D- and L- amino acids, or an arnide thereof, such as an amide
derived from ammonia, an ~1 -4C)a}kylamine (such as methylamine) or a di( 1 -4(::)al~cylamine
(such as dimethylarnine). A particular group of values for R4 includes, for exarnple, those
values defined herein where R4 is not a sequence of 1 to 6 arnino acids.
A ~l~fe..~,d value for R4 includes, for example, 4-carbamoyl-1-piperidyl (the residue
of piperidine-4-carboxamide (Pip-NH2)), 4-carboxy- 1 -piperidyl (the residue of piperidine-4-
carboxylic acid (Pip-OH)), 4-(carbamoylmethyl)anilino (the residue of 4-
aminophenyl~et~mide (Papa-NH2)), 4-(carboxymethyl)anilino (the residue of 4-
10 aminophenylacetic acid (Papa-OH)) and 4-(2-guanidinoethyl)anilino (the residue of 2-(4-
aminophenyl)ethylguanidine (Pape-NHC(=NH)NH2).
A particular group of values for R4 includes, for example~ Pip-NH2, Papa-NH2,
Pape-NHC(=NH)NH2 and N H Rc in which Rcis 3-guanidinopropyl, 2-morpholinoethyl or
4-(2-(2-hydroxyethoxy)ethyl- 1 -piperazinyl.
A preferred value for R2 includes, for example, a group of the forrnula II, especially IIa
and more especially IIb.
A preferred group of peptide derivatives of formula I includes, for example, peptide
derivatives in which Rlis a sequence of five L-arnino acids and R3is a single L-amino acid,
which sequence Rl and R3 have any of the values defined above, including the particular and
20 preferred values for R'and R3. Within this group, a sub-group of particularly preferred
peptide derivatives includes, for example, those in which R2 is a group of the formula II,
especially IIa and more especially IIb. A further sub-group of particularly plGfe.l~d peptide
derivatives includes, for exarnp}e, those in which R4is -Pip-OH, -Pip-NH2, -Papa-OH or
Papa-NH2. An especially preferred sub-group of compounds includes~ for example, those in
25 which R3 taken together with R4is Ala-Pip.N H2 or Ala-Papa-NH2.
A further preferred group of peptide derivatives of the invention include, for example,
those in which Riis a sequence of S L-amino acids lc;p~eselrted as AAl-AA2-AA3-AA4-AA5
in which:
AAl is selected from Ala, lle, Tyr, Val, Glu, Lys, Arg, Gly. Gap, GapMe4 and 3,3,3-
30 trifluoroalanine, particularly Ala, Ile, Arg, Gap and GapMe4~ especially Ala, Arg and GapMe4and more especially Ala and Arg;
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- 10 -
AA2 is selected from Ala, Lys, Glu, Sar, Val, Arg, Gly, Pro, Ile, Tic, 3,3,3-trifluoroalar~ine
and N6-dieth~lLys, particularly Ala, Arg, Ile, Lys and Tic, especially Ala, Arg, Lys and Ile
and more especially Ala and Arg;
AA3 is selected from Ala, His, Gln, Val, Thr, Glu, Gly, Asp, Asn and N3-diethylDap,
particularly Ala, His, Asp and Asn, especially Ala and Asn and more especially Ala;
AA4 is selected from Ala, Lys, Asn, Arg, Thr, Glu, Sar, Gly, Pro, His and N6-diethylLys,
particularly Ala, Arg, Lys and His, especially Ala~ Arg and His and more especially Ala; and
AAS is select~ d from Thr? Val, Ala, Gly, Dap, Dab, Pro, Hyp, Asn and N3-diethylDap,
particularly Thr, Val and Dap and especially Thr and Val; and
l O R3 is a single L-arnino acid selected from Ala, Gly, Dap, a7~ nine and azaglycine,
particularly Ala, Gly and azag~ycine, especially Ala and Gly and more especially Ala;
and wherein P, R2 and R4 have any of the values, including the particular and preferred values~
defined above. Within this group, a particular sub-group of compounds include. for exarnple~
those in which the sequence AA1-AA2-AA3-AA4-AA5 is selected from Ala-Ala-Ala-Lys-
Val, Ile-Ala-Ala-Arg-Th~ Arg-Ala-Ala-Ala-Val, Arg-Ala-Ala-Ala-Thr. Ala-Ala-Ala-Arg-
Val, Ala-Arg-Ala-Arg-Val, Ala-Ile-Ala-Arg-Val, Ala-Arg-Ala-~Iis-Val. Ala-Ala-Asn-Arg-
Val, Ala-Arg-Ala-Ala-Thr, Ala-Arg-Ala-Arg-Thr, Gap-Ala-Ala-Ala-Thr. GapMe4-Ala-Ala-
Ala-Thr and Ala-Ala-Ala-A}g-Thr. Compounds in which R4 is Pip-NH" Papa-~H2 and
Pape-NHC(=NH~NH2 are preferred.
A further preferred group of peptide derivatives of the invention include. for exarnple,
those in which R~ is a sequence of 3 L-amino acids represented as AA l -AA2-AA3 in which:
AAl is selected from Ala, Ile, Tyr, Val, Glu, Lys, Arg, Gly, Gap~ GapMe4 and 3.3,3-
trifluoro~i~nin~, particularly Ala, Ile, Arg, Gap and GapMe4, especially Ala. Ar~ and GapMe4
and mo}e especially Ala and Arg,
AA2 is selected f}om Ala, Lys, Glu, Sar, Val, Arg, Gly, Pro, Ile, Tic, 3.3,3-t}ifluoroalanine
and N6-diethylLys, particularly Ala, Arg, Ile, Lys and Tic, especially Ala, Arg, Lys and Ile
and more especially Ala and Arg;
AA3 is selected from Ala. His, Gln, Val, Thr, Glu, Gly, Asp, Asn and N~-dieth! lDap,
particularly Ala, His, Asp and Asn, especially Ala and Asn and more especially Ala; and
R3 is a se~uence of 3 L-arnino acids represented as AA6-AA7-AA8 in ~ hich:
AA6 is selected frorn Gly. Leu, Lys, Ala, Pro, Glu, Sar, His and Dap, particularl~ Ala. Leu
and Pro and especially Ala:
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- 11 -
AA7 is selected from Pro, Ala, Lys, Arg, Glu, Sar, Gly, Oic and Dic, especially Ala and Arg;
and
AA8 is selected from from Ala, Gly, Dap, ~7~ ninf and azaglycine, particularly Ala, Gly
and azaglycine and especially Ala; and P, R2 and R4 have any of the values, including the
S particular and ~lGr~,~,ed values, defined above. Within this group, a particular sub-group of
compounds incl~ s, for ~xan~lc, those compounds in which the sequence AAl-AA2-AA3 is
selected from Ala-Lys-Ala and Ala-Arg-Ala and the sequence AA6-AA7-AA8 is selected
from Ala-Ala-Ala and Ala-Arg-Ala. Compounds in which R4 is Pip-NH2, Papa-NH2 andPape-NHC(=NH)NH2 are preferred.
A ~ lcd aspect of the present invention comprises peptide derivatives of the
formula I, in which R2 is a group of the formula II (and especially wherein A is methylene),
and more especially IIb, and P, R', R3and R4 have any of the values, including the particular
and pler~ d values, defined above.
A further preferred aspect of the present invention comprises peptide derivatives of the
formula I which contain an arginine residue, particularly compounds in which the first amino
acid residue of Rl (as read from left to right) when it is a sequence of S L-amino acids is
arginine (such as Arg-Ala-Ala-Ala-Val and Arg-Ala-Ala-Ala-Thr) and compounds in which
the second amino acid residue of Rl when it is a sequence of 3 L-amino acids is arginine
and/or the second amino acid residue of R3 when it is a sequence of 3 L-amino acids is
arginine (such as when Rl is Arg-Ala-Ala and R2 is Ala-Ala-Ala or when Rl and R2 are both
Ala-Arg-Ala.)
A further aspect of the present invention comprises peptide derivativesof the
formula I in which R2 is a group of the forrnula IIIa or lIIb and P, Rl, R3 and R4 have any of
the values, including the particular and preferred values, decribed above.
ColllpoLulds of the invention which are of particular interest include, for exarnple, the
specific embodiments set out hereinafter in the acco,.~panying Examples. Of these, the
compounds of Examples 5, 16, 19, 23 and 24 are of special importance and these compounds,
or a ph~ e~ltically acceptable salt thereof, are provided as further features of the invention.
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-12-
(SEQ ID NO:S)
Phv-Arg-Ala-Ala-Ala-Vai-N~ N'Jl'Ala--N~CONH2
Example 5
(SEQ ID NO:17)
Me2N NMe2
Phv--N ~~CO -Ala-Ala-Ala-Thr-HN ~ ~N ~ J~~Aia--HN ~CH2CONH2
Exampie 16
CSEQ ID N 0:20)
Phv-Arg-Aia-Ala-Ala-Thr-N~ ~N'Jl'Gly--N ~ N~
Example 19
~SEQ ID NO:24)
Phv-Ala-Arg-Ala-N~ N'~~N-AIa-Arg-Ala-Hi~CH2CONH2
Example 23
-
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W 097131023 PCT/GB97100438
-13-
(SEQ: ID NO: 25)
CN
S~
,~,
co-A~a-Arg-Ala-N.~ NJ~'N-Ala-Arg-Ala-N~cH2coNH2
Example 24
Ph~rm~reutically acceptable salts include, for example, for peptide derivatives that are
sufficiently basic, for example those having a free amino group, salts with acids forrning
5 physiologically acceptable anions, such as salts with mineral acids, for example, hydrogen
halides (such as hydrogen chloride and hydrogen bromide), sulphonic and phosphonic acids,
and with organic acids such as acetic, oxalic, tartaric, m~n-lPlic7 p-tol~lenPe-7lrhonic,
meth~n~slllphonic acids. trifluoroacetic and the like, and for peptide derivatives that are
sufficiently acidic, for example those having a free carboxylic acid group, salts with bases
10 forrning physiologically acceptable cations, such as salts with alkali metal (such as sodium
and potassium), alkaline earth metal (such as m~gnPcium and calcium), aluminium and
arnmonium salts, as well as salts with suitable organic bases such as ethanolamine,
methylamine, diethylamine, isopropylamine, trimethylamine and the like.
As stated above, the peptide derivatives of formula I, or a ph~rrn~reutically acceptable
15 salt thereof, will have beneficial ph~rm~rQlogical effect in warm-blooded ~nim~le (including
man) in a range of autoimmune diseases or merlic~l conditions, to treat symptoms or as a
disease modifying agent or as a prophylactic tre~ment Such ~ cp~ees may include, for
example, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, idiopathic
- thrombocytopenic purpura, juvenile rhPllm~toid arthritis, coeliac disease, systemic lupus
20 erythemzltosllc, ankylosing spondylitis, Sjogren syndrome, my~etheni~ gravis, Type l (insulin
dependent~ diabetes, ~;~ehimoto7s disease, Grave's tli~e~ee, Addison's disease, scleroderma,
polymyositis, ~lPrmz~tl~myositis, pemphigus vulgaris, bullous pemphigoid~ autoimml-ne
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haemolytic ~n~n~i~, pernicious ~n~.~.mi~ glomerulon~phrit;~, graft rejections and such like,
especially rhPI~m~tQid arthritis and multiple sclerosis.
The utility of the peptide derivatives of the formula I, or a rh~rm~e--tically acceptable
salt thereof, may be ~ ecl using a variety of standard tests and clinical studies, including
those described in International Patent Application, Publication Nos. W092/02543,
WO93/05011 and W095/07707 (or modifications thereof~ and those described below. The
peptide derivatives of formula I show ~i~nifi~nt activity in one or more of such tests or
studies.
Test A: Purified HLA-DR peptide in vitro binding assay. (This assay may be used to
10 demonstrate the binding of the peptide derivatives of formula I to disease-associated MHC
class II molecules.) 30,u1 of biotin-FHA 307-320 (FHA (307-320) peptide. derivatised with long-
chain biotin at the N-tf~rminl~c, Biotin-Ahx-Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-
Ala-Thr-Gly-OH) at 800nM in phosphate buffered saline solution (PBS) is incubated with 30
~L of purified HLA-DR4Dw4 at a concentration between 0.5 and 5 ~g/ml in V-welled micro-
15 titre plates (Nunc) for 48 hours with or without inhibitor peptides. At the end of the
Incubation period 100~11 of the incubate is tr~ncferred to Enzyme Linked TmmllnoSorbant
Assay (ELISA) plates (Nunc) previously coated with an anti-MHC antibody (L243 -
American Type Culture Collection (ATCC) HB55 - as described in T ~mr~on and Levy (1980)
J Tmml-nol. 125,293-299)ataconcentrationof10~g/mlfor1houratroom~l~ ,dlureand
20 blocked thereafter for 1 hour with 1% bovine serum albumin (BSA) in PBS and 0.05% Tween
20. After a further 1 hour period the unbound peptide is washed away and a 1/4,000 dilution
of streptavidin peroxidase (Sigma) in PBS with 0.01% of a suitable deLelgellt such as NP-40
(Sigma) added for 2 hours at room telll~aLL~e. After further washing, tetramethylbenzidene
(TMB) s~lbstr~te solution (1 TMB tablet (Sigma~ in 10 mls of 0. lM citrate/acetate buffer, pH
25 6.0 with 36 ,~LI urea hydrogen peroxide (UHPO) (Fluka)) is added to each of the plates. The
reaction is stopped by adding :2M sulphuric acid (10~1 per well) and the absoll~,l-.ce read at
450nrn to quantify the arnount of peptide bound. The inhibitory activity of peptides is
obtained by plotting absorbance against concentration.
The purified HLA-DR4Dw4 may be obtained as follows:-
30 ~1) Expression of HLA-DR in the Baculovirus system
The expression of recombinant proteins in insect cells from baculovirus vectors is an
established procedure to obtain high yields of recombinant protein [Luckow7 VA & Summers,
,
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i~D, 1988, Biotechnology, 6~ 47-551 ~. To enable the c;~ ion of the heterodimeric HLA-
DR, eg. HLA-DR4Dw~, from a single recombinant baculovirus vector (as opposed to having
separate recombinant viruses for the a and ~ chains and then doing a co-infection), a double-
recombinant baculovirus is constructed which carries both the a and ,B chains.
A cDNA encoding the sequence of the a polypeptide is cloned into the transfer vector
pacYMl ['~ t~ullr~ Y; Possee, RD; Overton, HA & Bishop, DHL, 1987, J. Gen. Virol.,
1233-1250] to place e~ ,s~ion of the protein under the control of the polyhedrin promoter.
The unit is inserted into the baculovirus genome by homologous recombination in Sf21 insect
cells to create a single recombinant baculovirus for the a chain. The techniques for the
culture and infection of insect cells, for the homologous recombination and detection/isolation
of recombinant viruses are all fully described by Summers, l~DD & Smith GE (1987) [A
Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures; Texas
Agricultural Experiment Station, Bulletin No. 1555]. The molecular genetic techniques used
to construct the recombinant vectors are likewise readily available in the literature and are
most fully described by Sambrook, J; Fritsch, EF & Maniatis T, (1989) [Molecular Cloning.
A Laboratory Manual. 2nd F~liti~-n Cold Spring Harbor Laboratory Press].
To create the double-recombinant baculovirus, a cDNA encoding the ~ chain is cloned
into the transfer vector pAcUW1 rWeyer, IJ; Knight, S & Possee, RD. 1990, J. Gen. Virol.,
71, 1525- 1534] to place expression of the protein under the control of the P 10 promoter. The
unit is then inserted into the genome of the single recombinant baculovirus carrying the a
chain. Double-recombinant viruses are d~tect~d by spotting insect cells, infected with
randomly picked viruses from the transfection, onto membranes and reacting them with a
monoclonal antibody, e.g. L243, which specific~lly recognises the HLA-DR heterodimer.
Binding of the antibody to Sf21 insect cells is det~ct~ d using standard flow cytometry
techniques, readily available in the li~.dlulc Stable, double-}ecombinant baculovirus
C2~ CSsillg HLA-DR are plaque-purified.
(ii) Purification of HLA-DR from insect cells
- The method used is a modification of that described by Gorga et al 1987. (Gorga et al 1987.
J. Biol. Chem. 262, 1 ~087- 16094). HLA-DR expressing baculovirus/Sf21 cells (1 OL which is
approximately equal to 2 x 10 lO cells) are solubilised in lOOrnl of 5mM EDTA (sodium salt),
50mM Tris-HCL pH 8.5. 2% NP40. 150nM NaCl, lmM iodo~cet~mide, lmM PMSF by
homogenisation with 10 strokes of a teflon glass homogeniser. The homogenate is spun at
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1 OO,OOOg for 1 hour and the C~lp~ t~nt collected. The anti-HLA-DR monoclonal antibody
LB3.1 (Gorga et al 1986, Cell. ~mm-ln~l. 103, 160- 172) covalently coupled at a ratio of 50mg
of L243 to lOml of Protein A-Sepharose fast flow (Ph~rm~ci~) and pre-inr-lk~t~d with lOmM
Tris-HCl, pH 8.0, 0.1% NP-40 is inc~llh~fed overnight with the supern~tAnt The resin is then
put into a colurnn and washed with 1 OmM Tris-HCl, pH 8.0, O. I % NP-40 (20 colurnn
volumes) followed by 0.15 M NaCl, 50nM Na2HPO4, pH 7.0 1% octylglucoside (20 column
volumes). The HLA-DR is eluted with 50mM diethylamine pH 11.0, O.15 M NaCl, I %
octylglucoside. Colurnn fractions are imme~ t~ly neutralised with I M Tris-HCl pH 8.0 and
concentrated by ulllac~ ;fugation through a cenkicon-1O membrane. Protein content is
10 determined by a BCA protein assay (Pierce) and purity by SDA-PAGE electrophoresis.
In general, the peptide derivatives of formula I as defined above which were tested in
test A showed .ci~nifi~ nt inhibition at a concentration of about l O,uM or much less.
A further preferred aspect of the present invention comprises a peptide derivative of
the formula I, or a pharTnaceutically acceptable salt thereof, which does not bind to HLA-
15 DR3 but binds to HLA-DR1 and/or HLA-DR4Dw4 and/or HLA-DR4Dw14. HLA-DR3 is a
common HLA-DR allele which is not associated with rh~un~toid arthritis. Accordingly, in
rh.-llm~tr~id ~LhLilis patients who carry HLA-DR3 as one oftheir alleles (which is
approximately one third of the total rh~um~tf-id arthritis patients), such a peptide derivative of
the formula I will not interfere with the normal role of HLA-DR3 in the host-defense function.
The use of such a peptide derivative is therefore particularly advantageous for treating
rheumatoid arthritis patients as it will result in less immlmosuppressjon than would occur with
a non-selective DR binder.
As a variant to test A, the ability of a peptide of the invention to bind to one or more
HLA-DR molecules was ~ssec~e~ as follows:
(i) P~ ific~tion of HLA-DR Types from Cell lines
The method used w~ a modification of that described by Gorga et al, 1987, J.Biol.Chem.
262, 16087-16094. Human HLA-DR antigens were purified from various cell lines byimml-n~ffinity chromatography. Briefly, lxlO9 - 5 x 109 pelleted cells ofthe appropriate cell
line selected from Hom 2 (source of DR1), BBF (source of DR2), AVL-B (source of DR3),
JAH (source of DR4Dw4), JHAF (source of DR4Dw13) or PE117 (source of DR4Dw14) were
solubilised at approximately 4~C ~n 50 ml of 5 mM EDTA (sodium salt), 50 mM Tris-HCL
pH 7.4, 2% NP40, 150 mM NaCl, 1 mM iodoacetamide, I mM PMSF7 by homogenisation
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with 10 strokes of a teflon glass homogeniser. The homogenate was spun at 100,000 g for 1
hour and the sUpern~t~nt collected. The anti-HLA-DR monoclonal antibody LB3.1 (Gorga et
al, 1986, Cell Tmmlmol, 103, 160-173) covalently coupled to CNBr-Sepharose 4B
(ph~ ) was pre-e<~uilibrated with 150 mM NaCI, 50 mM Tris-HCL, pH 7.4, 0.1% NP-40
and incubated overnight with the sllpçrn~t~n1 The resin was then packed in a column and
washed with 0.15 M NaCl, 50 mM Tris-HCL, pH 7.4, 1 % octylglucoside (20 column
volumes). The HLA-DR was eluted with 50 mM diethylamine pH 11.0, 0.15 M NaCI, 1 %
octylglucoside. Column fractions were immediately neutralised with 0.5 M HEPES NaOH
pH 7.4. Protein content was ~let~rmined by a Biorad protein assay and purity by SDS-PAGE
10 clc~i~uphoresis.
(ii) Peptide Selectivit~ Bindin~ Assays
200nM biotin-FHA 307-320 in phosphate buffered saline (PBS) was incubated with either
purified HLA-DRl~ DR2, DR4Dw4, DR4Dw13 or DR4Dw14. (2-2011g/ml) in V-well
microtitre plates (Nunc) with or without inhibitor peptides in assay buffer (PBS, 0.01% NP40
15 (Sigma.)) For DR3 inhibition, 400nM Biotin-Ahx-(D)Ala-Ala-Ala-Che-Ile-Ala-Ala-Ala-Thr-
Leu-Lys-Ala-Ala-(D)Ala-OH was incub~ted with purified DR3 (20,ug/ml.), and incubated as
above. After 48 hours, the in.;ul,~Les were treated, and absorbance readings taken as described
in Test A. The inhibitory activity of peptides, ~x~rtssed as IC50 values, was calculated using
Microcal Origin software on a PC.
Test B: Inhibition of T cell activation in vitro. (This assay may be used to demonstrate the
ability of the peptide derivatives of formula I to inhibit a T cell immllnF~ response mediated by
or through an MHC class II molecule).
~nhibitor peptides were tested for the ability to block stimulation of the E~52.24 murine
25 T cell hybridoma line which responds to the FHA307320 peptide (H-Pro-Lys-Tyr-Val-Lys-Gln-
Asn-Thr-Leu-Lys-Leu-Ala-Thr-Gly-OH) ~ senl~d by HLA-DR4Dw4 molecules. B52.24
was produced by the fusion of Iymph node T cells taken from FHA30,.320 immllni~ed HLA-
DR4Dw4 ~nsg~llic mice (International Patent Application, Publication No W095/03331)
with the BW5147 murine T ceII lymphoma line (White et al (1989) J. Immunol. 143, 1822) as
outlined in Woods et al (1994) J. Exp. Med. 180, 173-181 and following the general methods
for the generation of T cell hybridomas given in Current Protocols in Immunology, Volume 2,
7.21.
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Inhibitor peptides at col-c~ . alions b~L~ 100 and O.l ,uM (or lower) were mixedwith the antigenic peptide FHA307 320 in either varying concentrations between 100 and 0.1 11
M or at a fixed concentration of 10 ,uM by dilution in RPMl-1640 culture media (Gibco) in a
96-well mictrotitre plate (Nunc) in a final volume of 100 ~Ll. E~LA-DR4Dw4 e~le~ g B
S cells such as the JAH E~BV l~ ed lymphoblastoid cell line (European Culture
Collection ECACC 85102909) or B cells taken from an HLA-DR4Dw4 homozygous
individual and transfontned with Epsten Barr virus according to tne method described in
Current Protocols in Tmmllnology 7.22.1 were fixed using gluteraldehyde by suspension in
1~/~ gluteraldehyde (Sigma) at 4 x I o6 cells/ml for 30 seconds, after which an equal volume of
10 200mM Iysine (Sigma) was added for 3 minut~ The cells were recovered by centrifugation
at 300g, washed in RPMI-1640 and added to the microtitre plates cont~inin~ antigen and
inhibitor compounds at a conce~ dLion of 2 x 105 cells per well. The microtitre plates were
incubated for 2 hours at 37~C and 5% CO2.
The microtitre plates were then washed in RPMI-1640 by centrifugation at 300g and
15 aspirated twice before the addition of the B52.24 T cell hybridoma line at a concentration of
105 cells per well in culture medium (RPMI- 1640, 10% foetal calf serum (Gibco) and 2mM
glutamine ~Gibco)). The microtitre plates were then incubated for a further 2 days at 37~C
and 5% CO2. The plates were then centrifuged at 300g for 10 minutes and 1 501l1 of
supernatant removed from all wells to be frozen at -20~C prior to bioassay for IL-2 content.
The culture plates conf~inin~ sUp~rns~t~ntc to be assayed were left at room telllpe.dLIlre
to thaw and l OOml of ~u,~ nt was transferred to fresh 96 round bottomed well plates. 1:1
serial dilutions of IL-2 were carried out using culture media (RPMI- 1640 (Gibco), 10% foetal
calf serum (Advanced Protein Products), 100 !lg/ml streptomycin and 100 U/ml penicillin
(Gibco), 2mM L-pl~lt~mine (Gibco) and 50 ~lM 2-mercaptoethanol (Sigma)), to produce a
25 standard curve of 250 units/ml to 0.04 ur~its/ml IL-2 final. An IL-2 dependent cell line such
as CTI.L-2 cells (Nature (l977) 268 154-156) or HT-2 cells (J. Tmmllnol Methods (1987) 94-
104) were harvested and washed twice using culture media prior to resuspension at 5 x 104
cells/ml. I OO,ul of IL-2 dependent cell suspension was added to each well of the standard
curve and test samples. The culture plates were in~uh~t~A for 72hrs at 37~C and 5% CO2.
30 After which, 20~ I rnCi) of 3H-Thymidine (Amersham International) was added to each well
and the plates returned to the incubator for a further 1 6hrs. The contents of each plate were
.~
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harvested onto glass fibre filter mats and the radioactivity measured using a betaplate
scintill~tion counter.
In general, the peptide derivatives of formula I as defined above which were tested in
test B showed significant inhibition at a concentration of about 1 0,uM or much less.
S
Test C: Peptide stim~ t~cl DTH (delayed type hypersensitivity) in BALB/C mice. (The
assay may be used to demonstrate in vivo activity of peptide derivativesof formula I in an
animal model). Balb/c female mice (18-20g), 5 per group, were immuni~e-l sub-cutaneously
on the flank with O.lml of an emulsion of ovalbumin (Sigma) (2mg/ml in saline) mixed 1:1
10 (v/v) with complete Freunds adjurant (Sigma). Seven days later footpad thicknPcc was
~iet~min~cl using a dual caliper micrometer followed by a challenge in one hind footpad with a
30~Ll sub-plantar injection of 1% heat-aggregated ovalbumin protein in saline. Twenty-four
hours after antigen challenge, footpads were measured and the DTH response calculated as the
percentage increase in footpad thickness in the injected footpad compared to contralateral
15 control. Inhibitors were ~-lmin~-red by 3-day osmotic mini-pumps (Alzet) implanted 24
hours prior to antigen challenge at doses ranging from 1 Omg/kg/da! to 0.1 ~g/kgtday. The
degree of inhibition was calculated by substracting the value for s~-elling of inhibitor treated
footpads from that of the vehicle dosed controls, dividing by the control value and multiplying
by 100%.
In general, the peptide derivatives of formula 1 as defined above which ~vere tested in
Test C showed significant inhibition at a dose of about 1 mg/kg/da~ or much less. without any
overt toxicological or other ullLow2lld ph~ cological effect.
Test D: (This assay may be used to demonstrate in vivo activity of peptide deri~ atives of
formula I in an animal model of arthritis).
Balb/c female mice (19-21g, 5-10/group) are immuni~ed on day 0 and boosted on day
7 with a sub-cutaneous injection of 0. lml of an emulsion cont~ining equal volumes of 2mg/ml
methylated bovine serum albumin ~met-BSA, Sigma) in saline and complete Freund'sadjuvant (Sigma) supplemented with 2.5mg/ml Mycobacterium tudercolosis (MTB, strains ~,
DT and PN, MAFF, Weybridge, Surrey) thus giving a final MTB concentration of 3.5 mg/ml.
An additional 0.1 ml i.p injection of 109 Bordetella pertussis or~ni~mc (Wellcome Pertussis
vaccine) in saline is given at the same time. Fourteen days later, ~nim~l~ are challenged into
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one knee joint with a 101l1 intra-articular injection co~ ,g lOOug of met-BSA in saline
using a 30G needle and hamilton syringe. The contralateral knee is injected with a similar
volume of saline and serves as a control. The degree of infl~mm~tion/swelling associated
with both knees is ~Içtermin~ 13 days later by me:~ellring using a dual-caliper micrometer.
S This is achieved by making an incision with blunt-ended scissors and forceps into the skin
approximately Srnm above and below the knee and co..l illll;l~g along the side of the knee to
form a flap which is then carefully cut away to expose the underlying joint. Measurements
are made across the widest part of the knee, in the ho~i ~onl~l plane, on the flexed limb held in
a fixed position. Percentage increase in infl~mm~f;on in the antigen-inJected knee co~l,paled
to control is calculated according to the formula: [antigen-injected knee thickness - saline-
injected knee thickness/saline-injected knee thickness] x 100. Inhibitors are ~Aminict~red
using 14 day osmotic mini-pumps (Alzet) impl~ntefl 24 hrs before antigen challenge at does
ranging from lOmg/kg/day to O.lug/kg/day. The ~cleelll~ge inhibition of
infl~rnmAtion/swelling is calculated from the thickness mea.-uG~l~ents by subtracting the value
for swelling in the inhibitor-treated group from that of the vehicle dosed controls, dividing by
the control value and multiplying by 100. Additional ~ses~mPnt~ of disease involve 1) the
histological evaluation of inll~mm~tion, synovitis and cartilage/bone erosions carried out on
fixed knee sections stained with haemotoxylin and eosin and 2) the determination of levels of
acute phase reactants in serum, serurn amyloid P and/or haptoglobin.
Peptide derivatives of forrnula I as defined above may show in Test D significant
inhibition at a dose of about 10 mg/kg/day or much less.
By way of illustration of the ph~ Acological activity of particular peptide derivatives
of the for}nula I, the compounds of Examples 5, 16 and 23 all showed significant binding to
HLA-DR4Dw4 in Test A at a concentr~ion of <0.1 micromolar, and were active at <0.1
mg/kg/day in Test C. These compounds also showed good aqueous stability at pH3 and pH
7.6 and, in the form of an extruded polymer depot formul~tion, showed minim~l loss due to
degradation on extrusion and minim:~l degradation on release from such a depot formulation.
In the variant to Test A, the compound of Example 23 was shown to bind significantly to
HLA-DR1, HLA-DR2, HLA-DR4Dw4 and HLA-DR4Dw14 but did not bind significantly to
HLA-DR3 (IC~o > 100 micromolar).
A peptide derivative of formula I may be prepared by any process well known in the~
art of peptide chemistry to be applicable to the synthesis of analogous peptides.
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A peptide derivative of formula I may be obtained, for example, by procedures
analogous to those disclosed in "Solid Phase Peptide Synthesis: A practical approach" by
Atherton and Sheppard (published by IRL press at Oxford University Press, 1989). "Solid
Phase Peptide Synthesis" by Stewart and Young (published by the Pierce Chemical Company,
Illinois, 1984), "Principles of Peptide Synthesis" (published by Springer-Verlag, Berlin,
1984), and a series of books "Amino Acids, Peptides and Proteins" (volumes 1 - 25; volume
2~ published in 1994~ (published by the Royal Society of Chemistry, Cambridge, UK).
Preferably, a peptide derivative of formula I is prepared by solid phase se~uential
synthesis. Using this technique, the amino acid which is to become the C-t~rminn~ arnino
10 acid of the peptide is protected at the alpha-amino group, and, if necessary, in the side chain
and coupled to a solid support, for exarnple a resin~ such as 2-chlorotritylchloride resin or
Merrifield resin (chloromethylpolystyrene-divinylbenzene) if a free carboxylic acid is
re~uired after cleavage, or Rink Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxy resin) or Rink Amide MBHA resin (N-(4-(2',4'-dimethoxyphenyl-Fmoc-
15 aminomethyl)-phenoxy~ret~mido-norleucyl)-4-methyl benzhydrylamine resin) (all available
from Calbiochem-Novabiochem) if a carboxamide is required after cleavage, whereafter the
protecting group on the alpha-arnino group is removed. The arnino acid which is to be
attached to the C-terminl-~ amino acid is protected at the alpha-amino group and~ if necessary.
in the side chain and coupled to the C-t~.rrninl~ amino acid which remains attached to the
20 solid support. The stepwise process of deprotection of the alpha-amino group and coupling to
the next arnino acid is repeated to give a protected or unprotected polypeptide attached to the
solid support. The group R2 of formula II or III is incorporated into the sequence by using an
appropriately protected (3-amino-2-oxo-pyrrolidin-1 -yl)alkanoic acid (for a peptide derivative
cont~inin~ II in which A = methylene) or a corresponding oxa analogue obtained as described
in ~. Med. Chem., 1993, 36, 256-263 or by analogy therewith (for a peptide derivative
cont~ining II in which A is oxygen) or an (6-oxo-l~7-dia~s~ o[4.4]non-7-yl)alkanoic acid
(for a peptide derivative cont~inin~ III) in place of a protected amino acid. The protected or
unprotected polypeptide is released from the solid support by standard procedures, for
example using a mixture of trifluoroacetic acid, triethylsilane and water. It will be
appreciated that a side-chain protecting group may be cleaved under the conditions used to
release the peptide from the solid support. or may be cleaved as a separate step prior or
subsequent to release of the peptide from the solid support. It will also be appreciated that the
CA 02242809 1998-07-09
W O 97/31023 PCT/GB97/00438 -22-
procedure to build up the polypeptide may be modified by using a sequence of two or more
suitably protected amino acids in a particular coupling step. The synthesis may use manual
techniques or be carried out ~ntom~tically, employing for exarnple, an Applied Biosystems
431A or 430A peptide synth~si~t-r, an Advanced Chemtech ACT357 peptide synthesiser or
5 similar ~ o~ "~ ic peptide synthesiser, or a combination of both techniyues may be used.
During the assembly of the peptides, the amino acid functional groups not taking part
in the reaction are protected by various functional groups. For example, the N-t.-rmin~l and
side chain amino groups may be protected by using 9-fluorenylmethoxycarbonyl (Fmoc), t-
butoxycarbonyl (Boc?, biphenylisopropoxycarbonyl (Bpoc3, 2-[3,5-dimethoxyphenyl~propyl-
10 2-oxycarbonyl (Ddz), ~ m~ntyloxycarbonyl ~Adoc), allyloxycarbonyl (Aloc), 2,2~-
trichloroethoxycarbonyl ~Troc), benzyloxycarbonyl and various substituted
benzyloxycarbonyl groups. These protecting groups can be cleaved when required by the
standard techniques (e.g. acid or base treatment, catalytic hydrogenolvsis and Pd(0) treatment
or zinc/acetic acid trt~tment).
Suitable protecting groups used for the protection of the side chain guanidino group in
the peptides co~ irlg an arginine residue include a nitro, ~ m~n~yloxycarbonyl, 4-
methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), 2,2,5,7,8-pentamethvlchroman-6-sulphonyl
(Pmc) and ~especially) 2,2,4,6,7-pentarnethyldihydrobenzofuran-5-sulphonyl (Pbf) ~roup.
Suitable protecting groups used for the protection of a side chain h~-droxy group
20 include t-butyl, benzyl and trityl (Trt). Suitable protecting groups used for the side chain
imi{l~7ole group in the peptides cu.,l;.;.~;..g a hi.cticiine residue include a trityl, benzyl, tosyl,
dinitrophenyl, Adoc, Boc or Fmoc group.
Suitable protecting groups used for the protection of a side chain carboxyl group
include various esters (e.g. methyl, ethyl, t-butyl, benzyl, nitrobenzyl, allyl and 9-
2~ fluorenylmethyl).
The protecting group cleavage reactions can be perforrned at temperatures in the rangeof 4~C to 40~C (preferably at or about ambient temperature) and over a period of time in the
range of 10 minllt.oc to 24 hours.
Suitable coupling methods used for the coupling of the individual amino acids include
30 the commonly used a~ide, symmetrical anhydride, mixed anhvdride and various active esters
and carbodiimides. In the case of various carbodiimides (e.g. dicyclohex~ or diisopropyl-
carbodiimides), a number of additives (e.g. l-hydroxybenzotriazole (HOBT~ and N-
CA 02242809 1998-07-09
W 097/31023 PCTIGB97/00438
-23-
hydroxysllccinimde) may also be added. In addition, the amino acid couplings can also be
achieved by using a number of other reagents, e.g. l H-benzotriazole- 1 -yl-oxy-tris-
pyrrolidinophosphonium hexafluororh~sph~te (PyBOP), (2-(1 H-benzotriazole- 1 -yl)- 1,1,3 ,3 -
tetram~lhyl~uonium Lelldnlloroborate ~I'BTU) and (2-(lH-benzotriazole-l-yl)-1,1,3,3-
5 tetrarnethyluronium tetrafluoroborate (HBTU). The coupling reactions can be p)~, .Çolllled att~ ,.l...G:j in .tk~e ra.nge of -2Q~C ~nd ~L0~C and over a period of ~le in ~e range of 10
minl~t~s to 24 hours. A suitable me~ m for carrying out the coupling reactions includes, for
exa_ple, N,N-dimethylformatnide (DMF). A particularly suitable method includes the use of
HBTU, HOBT and diisop-~Jpylethylamine in DMF.
These and other methods of peptide synthesis are exemplified in the International
Patent Applications referred to herein. A hydrophobic residue P which is a group of the
formula R-, ~.CO-, R.SO2-, R.O.CO-, R.NHCO-, R.O.CS-, R.S.CO-, R.NHCS-, R.S.CS- and
R.CS- (or such a group present as a substituent on a terrninal amino group of P where P is a
hydrophobic amino acid or a hydrophobic amino acid bearing further amino acids) may be
15 incorporated, for example, as a final step by alkylation, acylation or other standard functional
group modification of a t~rmin~l arnino group (for example prior to or subsequent to release
of the peptide from a support). When C-t~ minl-c modifications are required (to obtain a
particular value for R4), they may be performed after the peptide is synthesised, by
conventional functional group modification or ap~,o~Liate choice of the initial starting resin or
20 the protected entity first coupled to the resin (for example by using a suitably protected group
of the formula R4-H). Typical exarnples of the ~l~,pa,cLlion of peptide derivatives of formula I
are provided in the examples hereinafter.
A typical yl~Jcedul~ for measuring the stability of a peptide derivative of the present
invention is as follows, in which, to minimi7t? microbial cont~min~tion and degradation, all
25 equipment that is used to prepare peptide solutions is sterilized in an autoclave and all
material transfers carried out in a Class II laminar flow cabinet. Approximately 20ml of
McIlvaine's citric acid-phosphate buffer solution at pH 3 or 7.6 cont~ining 0.02% sodium
a2ide is filtered into a 50ml bottle using a sterile 0.22,um filter unit and a 20ml syringe.
App~ "ately 1.2 mg of peptide is accurately weighed in a capped vial. Using a sterile
30 pipette tip, sufficient buffer solution is added to the peptide in the vial to give a peptide
concentration of 0.1 mg/ml. The vial is capped and shaken to dissolve the peptide. Using a
sterile pipette tip, aliquots of approximately l ml of the peptide solution are transferred to l 0
-
CA 02242809 1998-07-09
W O 97131023 ~CTIGB97100~38
-24-
HPLC vials, which are then capped. S vials are stored at -18 and 37~C. The area of the
peptide peak for the solution is f1~ ed by HPLC using al~ro~l;ate standards initially and
after storage at -18 and 37~C for 1, 2, 3 and 4 weeks, using a fresh vial at each time point with
duplicate sample injections. The pe.~;cllL~ge of peptide rem~ining after storage at 37~C at each
time point is de~rrn;ned from the ratio of the area o~the peptide peak at each time point to the
initial area. Preferred peptide derivatives of the present invention have greater than 90%, and
preferably greater than 95%, of peptide rem~;ning after storage at 37~C at both pH 3 and 7.6.
The peptide derivative of formula I will generally be ~lm;n;ct~red for therapeutic or
prophylactic purposes to warm-blooded animals (including man) requiring such treatment in
the for~n of a ph~rmslreutical composition, as is well known in the phz~rm~ellt;cal art.
According to a further feature of the invention there is provided a pharmaceutical
composition which comprises a peptide derivative of the formula I. or a pharm~reut;cally
acceptable salt thereof~ in association with a ph~rm~ceutir.~lly acceptable diluent or carrier.
The composition may be in a form suitable for oral use, for example a tablet, capsule~
aqueous or oily solution, suspension or emulsion: for nasal use, for exarnple a snuff, nasal
spray or nasal drops; for vaginal or rectal use, for example a suppository; for a~m;n;~tration
by inhalation, for example as a finely divided powder or a liquid aerosol; for sub-lingual or
buccal use, for example a tablet or capsule; or for parenteral use (including intravenous,
subcutaneous, intramuscular, intravascular or infusion~, for example a sterile aqueous or oily
solution or suspension. The composition may be in a form suitable for topical z~.1m;n;.~tration
such as for exatnple creams~ ointment~ and gels. Skin patches are also contemplated.
Formulation in general is described in Chapter 25.2 of Comprehensive Medicinal Chemistry,
Volume 5, Editor Hansch et al, Pergamon Press 1990.
In general the above compositions may be prepared in a conventional manner usingconventional excipients. However, in the case of a composition for oral a~mini~tration, it
may be convenient for the composition to include a coating to protect the polypeptide active
ingredient from the actions of enzymes in the stomach.
A preferred composition of the invention is one suitable for oral ~r~minstration in unit
dosage for~n for example a tablet or capsule which contains from 2.5 to 500mg, and preferably
10 to 100 mg, of polypeptide in each unit dose~ or one suitable for parenteral ~-lmini~tration
which contains from 0.5 to TOOmg of polypeptide per ml. and preferably 1 to I Omg of
polypeptide per ml of solution.
~ .,
CA 02242809 1998-07-09
W O 97/31023 PCTIGB97/OQ438
-25-
A pa~ ldl composition is pre~erably a solution in isotonic saline or isotonic dextrose
buffered if necessary to a pH of 5 to 9. ~It~rn~ively, the p~ al composition may be one
designed for slow release in which case the arnount of polypeptide per unit dose is in general
greater than that required when a collvelllional injectable forrnulation is used. A preferred
5 slow release formulation is a continuous release form~ tion, for exarnple a f~)rmlll~tion of the
type descri~ed in European Patent Application No. 58481 or, for peptides of formula I
cont~ining at least one basic group, a forrnulation as described in International Patent
Application, Publication No. W093/24150, which patent applications are incorporated herein
by reference. ~ertain peptides of the present invention possess solubility char~ctt~ri~tics
10 which make them particularly suitable for the m~mlf~tllre and proce~ing of slow release
parenteral formulations, particularly formulations cont~ining biodegradeable polyesters such
as polylactides. and for providing slow release formulations with beneficial release profiles.
Furthermore. peptides of the present invention cont~ining one or more basic groups,
particularly arginine, can also forrn peptide-polymer salts with acid-ended polyesters~ such as
15 polylactides, and such peptides and peptide-polymer salts con~LiLu~e a further aspect of the
present invention. Certain such salts possess solubility char~ct~ri~tics which make them
particularly suitable for the m~nllf~f~tl-re and proc~c~ing of slow release ~nLe~al
formulations, for example as described in W093/24150, and for providing slow release
formulations with beneficial release profiles and storage stability characteristics. A pl~re~lc;d
20 slow release parenteral formulation contains from l to lO0 mg (such as 5 to 50 mg) of
polypeptide per unit dose. A preferred slow release p~e.llelal formulation is also one
designed for slow release over a period of at least 5 days.
Preferred peptides of the present invention include those which, when in the form of
an extruded polymer depot formulation, show minim~l loss due to degradation on extrusion or
25 which show minimzll degradation on release from such a depot formulation. Typical
procedures for measuring the level of degradation of a peptide of the present invention are as
follows:-
Pl~u~dtion of extruded polvmer depot formulation of peptide
About 20 mg of peptide is accurately weighed and sufficient polymer (50/50% molar
30 poly(D,L-lactic acid/glycolic acid) copolymer of approximate weight average molecular
weight 20kD and approximate polydispersity of 1.7 as determined by size exclusion
chromatography relative to polystyrene standards) added to produce an approximate ~'0% w/w
CA 02242809 1998-07-09
W 097131023 PCT/GB97/00438
-26-
mixture. This is dissolved in anhydride-free glacial acetic acid to produce an approximate
10% w/v solution. The solution is freeze dried and the resulting freeze dried product is stored
under vacuurn prior to use.
S About 100mg of freeze dried m~t.o.ri~l iS loaded into the barrel of a small laboratory extruder
and the plunger pushed down to consolidate the sample. The extruder is heated to between 90
and 95 ~C and held at this temperature for 10 minutes before the freeze dried material is
extruded under pressure to give a cylindrical extrudate of a~ xilllately I mm in c~i~m~t~r.
10 Anal~sis of Peptide content of extruded PolYmer del~ot form~ tion of peptide
Two approximate Smm lengths of extruded polymer depot cont5~ining peptide are accurately
weighed and each dissolved in lml of anhydride-free glacial acetic acid in separate 25ml
volurnetric flasks. After about l .S hours the volume of each is made up to 25ml with distilled
water, causing the polymer to precipitate. The solids are filtered offusing a O.S~lm Millex
15 PTFE filter and the solutions, A, collected.
A series of standard solutions are prepared from a stock solution of peptide in distilled water
at O.Smg/ml and a stoclc solution of polymer in anhydride-free glacial acetic acid at 2.5mg/ml
as follows with each solutions made up to 1 Oml with distilled water:
-Concentration Volume of Volume of
of peptide(~Lstock polymerstock peptide
g/ml) solution (,u13solution (~1)
1000 1000
1 000 800
1000 600
1000 400
I O I 000 200
S 1000 100
O 1000 o
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W O 97/31023 PCTIGB97tOO438 -27-
Each standard is filtered through a 5~Lm Millex PT~E filter and an aliquot of filtrate, together
with aliquots of the solutions A, analysed by HPLC using duplicate sample injections. The
peptide content of the extruded polymer depot forrnulation of peptide is calculated from the
concentration of peptide in solutions A, which is cl~t~ d by COI~lp~ g the area of the
5 peptide peak in solutions A with the area of the peptide peak from the standard solutions.
Preferred peptides of the present invention show minim~l loss due to degradation on extrusion
and thus the peptide content of the extruded polymer depot formulation is close to the
approximate theoretical value of 20% w/w.
10 De,~radation of ~eptide on in vitro release from an extruded pol~mer depot
A solution of McIlvaine's citric acid-phosphate buffer solution at pH 7.6 con~ining 0.02%
sodium azide, is filtered through a 0.22~ filter and stored at 4~C. Approximately 10 mg of
extruded polymer depot cont~ining peptide is placed in two small vials and 2ml of the buffer
solution added. The vials are then capped and stored in a water bath at 37~C for a month. At
15 suitable time points over a month, three 0.6ml aliquots of release medium are removed from
each vial and either analysed by HPLC or stored frozen in an HPLC vial at -1 8~C prior to
ana}ysis by HPLC. 1 .8ml of the buffer solution is added to each vial con~inin~ the depot to
replace the release medium that has been removed at each time point.
20 The average amount of intact peptide in the release me~ m at each time point is determined
by HPLC using duplicate sample injections by CO~ dl;llg the area of the peptide peak in the
release media with the area of the peptide peak from standard buffer solutions of peptide at
known concentrations. The a~o~h~ate average arnount of peptide degradation products in
the release media at each time point is ll~tertnined by HPLC by comparing the area of
25 additional new peaks in the release media with the area of the peptide peak from standard
buffer solutions of peptide at known concentrations and ~ illg the extinction coef~lcient
has not changed. The average cumulative in vitro release profile of intact peptide and total
peptide ~intact peptide and peptide degradation products) is determined from the amounts of
intact peptide and peptide degradation products in the release medium at each time point.
30 Preferred peptides of the present invention show minim~l degradation on in vitro release and
thus show total peptide degradation products of less than 10% and preferably less than 5% of
CA 02242809 1998-07-09
W 097/31023 PCT/GB97/00438
-28-
total peptide after a month of in vitro release into McIlvaine's buffer solution at pH 7.6 at 37~
C.
The composition of the invention will generally be ~lnnini.~tPred to man such that, for
S example, a daily dose ~vill be from 10 micrograms to 5000~ng, preferably 0.1 to lOOmg, for a
70kg patient, given in divided doses as nccessa~ y. The precise arnount of composition
:~imini~tPred and the route and form of aclmini~tration may depend on the size, age and sex of
the person being treated and on ~he particular disease or mP~lic~l condition being treated and
its severity, according to principles well know in the mPtlic~l art.
A peptide derivative of formula I, or a ph:~rm~elltically acceptable salt thereof, may
also be advantageously ~lmini.ctered for therapeutic or prophylactic purposes together with
one or more other pharm~.ological agents known in the general art to be of value in treating
or relieving the symptoms of (or to be a disease modifying agent of) one or more of the
rii~e~es or mP-1ic~1 conditions referred to hereinabove, such as a NSAID (such as ibuprofen
15 or piroxicam), an analgesic (such as paracetamol), a corticosteroid, a muscle relaxant, a
lipoxygenase inhibitor, methotrexate, azathioprine, D-penicillQmine, Cyclosporin A or a
monoclonal antibody therapy (such as anti-CD4 or anti-TNF). In diabetes the peptide
derivative may be co-~-lminictPred with insulin or other therapies for diabetes or diabetes
complications, (such as an aldose reductase inhibitor). It is to be understood that such
20 combination therapy constitutes a further aspect of the invention.
According to a further aspect of the present invention there is provided a method for
treating a MHC class II dependent T-cell mPfli~t~rl autoimmunP or infl~mm~f-lry disease. for
example one or more of the diseases or medical conditions referred to herein, which
comprises ~rlmini~tering to a warm-blooded m~mm~l (including man) in need of such
25 tre~tmPnt an effective arnount of a peptide derivative of formula I, or a pharmaceutically
acceptable salt thereof. The invention also provides the use of a peptide derivative of formula
I, or a ~harm~f ellfically acceptable salt thereof, in the production of a novel medicament for
use in the treatment of a MHC class Il dependent T-cell me~ t~-l autoimmune or
irnflamm~t~-ry disease.
In addition to their aforesaid use in therapeutic medicine in hllm~n~ the peptide
derivatives of formula I are also useful in the veterinary treatment of similar conditions
affecting commercially valuable warm-blooded ~nimzll~ such as dogs, cats. horses and cattle.
CA 02242809 1998-07-09
W O 971310Z3 PCT/GB97100438
-29-
In general for such tr~tment, the peptide derivative of the formula I will be ~lmini.~l~red in
an analogous amount and manner to those described above for ~rlmini.~tration to humans. The
peptide derivatives of formula I are also of value as ph~rm~rological tools in the development
and standardisation of test systems for the evaluation of the effects of MHC class II molecules
in laboratory ~nim~l~ such as cats, dogs, rabbits, monkeys, rats and mice, as part of the
contint-in~ search for new and i~ v~;d thc~dyeuLic agents, or as diagnostic reagents.
The invention will now be illustrated by the following non-limiting Examples in
which, unless otherwise stated:-
(i) concentrations and evaporations were carried out by rotary evaporation in vacuo;
10 (ii) operations were carried out at room te~ lule, that is in the range 1 8-26~C;
(iii) yields, when given, are inten~1ed for the ~Csi.ct~nce of the reader only and are not
nececs~rily the maximum ~tt~in~hle by diligent process development:
(iv) the following abbreviations are used:
Phv = 5-phenylvaleryl; Boc = tert-butoxycarbonyl; tBu = tert-butyl; DMF = N,N-
15 dimethylro, ~ .i(ie; HOBT = l-hydroxy-bel~otliazole; Met = methionine; Fmoc = 9-
fluorenylmethyloxycarbonyl; Fmoc-Pip-OH = N-(9-fluorenylmethoxycarbonyl)piperidine-4-
carboxylic acid; Fmoc-Papa-OH = 4-rN-(g-fluorenylmethoxycarbonyl)amino]phenylacetic
acid; CbZ = benzylo~ycalbo-l~,rl; Pmc = 2,2,5,7,8-pentamethylchroman-6-sulphonyl; Pbf=
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl; THF = tetrahydrofuran; DMSO =
20 dirnethylsulfoxide; HBTU = 2-(lH-benzotriazol-lyl)-1,1,3,3-
tetramethyluronillmhc~11uorophosphate; DIPEA = diisopropylethylamine: TFA =
trifluoroacetic acid; Su is s-lccinimicle ~tt~r.hecl via the ring nitrogen atom; HPLC = high
pressure liquid chl~n~aL~graphy, and RP-HPLC = reverse phase high pressure liquid
chromatography (which unless otherwise stated was carried out on a V~ dac C 18 column
218TP54, 4.6 x 250 mm);
(v) flash chromatography and chromatography on silica were performed on Merck
Kieselgel 60 (Art No. 9385) obtained from E Merck, D~rm~t~(lt Gerrnany;
(vi) 'H NMR spectra were determined at 200 Mhz in CDCL3 or d6-dimethylsulphoxide(d6-DMSO) using tetramethylsilane (TMS) as an internal standard, and are expressed as
chemical shift (delta) values in parts per million relative to TMS using conventional
abbreviations for ciesi~n~tion of maJor peaks: s, singlet; m, multiplet, t~ triplet; br. broad. d.
doublet;
CA 02242809 1998-07-09
W O 97/31023 PCTIGB97/00438 -30-
(vii) the following Fmoc-protected amino acids were used for the introduction of a Lys,
Thr, Arg or His residue:
for Lys: Fmoc-Lys(Boc)-O~I; for Thr: Fmoc-Thr(OBu)-OH; for Arg: Fmoc-Arg(Pmc)-OH or
l:;moc-Arg(Pbf)-OH; and for H;s: Fmoc-His(Trt)-OH;
5 (viii) in Example 2, where formula III is referred to this means formula III in which Rb is
methyl;
(ix) in Examples 17 and 31, where in the name of the product the term 'morpholine' is
used, this means that the morpholine group is attached by the ring nitrogen to the adiacent
methylene group;
(x) in Example 18 and 34, where in the name of the product the terrn N(CH2CH2)2N' is
used, this 1~LCS~ ; a pi~ z.ne ring; and
~xi~ in Example 3û, where in the name of the product the term ~ -Lys(=C(NMe2)2-" is
used, this represents the L-amino acid residue -HN-CH[CH2CH2CH~CH~N--C(NMe2)2]-CO-.
-
CA 02242809 1998-07-09
W O 97/31023 PCT/GB97/00438 -31-
Example 1. Preparation of Phv-Ala-Ala-Ala-Lys-Val-IIb-Ala-Pip-NH2 (SEQ ID NO:l)
1.1 Synthesis of Boc-(D)-Met-(k)-Ala-OMe
S
~ O N 1~ O
S N-methylmorpholine (5.6g), k-alanine methyl ester hydrochloride (3.9g), HOBT (4.6g) and 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide (5.3g) was added to a solution of Boc-(D)-
Methionine (7g, 0.028mol) in dry DMF (SOml). The mixture was stirred overnight. Solvent
was removed by evaporation and the residue was partitioned between dich}oromethane
(lOOml) and 5% aqueous acetic acid (SOml). On ~t~n-lin~ HOBT crystallised and was
removed by filtration, and the organic layer was separated and washed with aqueous sodium
bicarbonate, dried (MgSO4) and evaporated. The residue (8.5g) was purified by flash
chromatography in a sinter funnel eluting with a mixture of dichloromethane and ether (0% to
100% ether). The fractions contS~ining product were combined and evaporated to give Boc-
(D)-Met-(O-Ala-OMe (7.2 g) as a gum which cryst~ ed on st~n-linf~; NMR (CDCl3): 1.4 (d,
3H), 1.45 (s, 9H), 1.95 (m, IH), 2.1 (s, 3H), 2.1 (m, lH), 2.6 (m, 2H), 3.75 (s, 3H), 4.3 (bs,
lH), 4.6 (m, IH), 5.3 (m, lH), 6.9 (bs~ lH).
1.2 S~ nthesis of methyl (2O-2-1(3B)-3-(~-[tert-butyloxycarbonyllamino)-2-oxo-
pyrrolidin-l -yllpropionate
~ ~ N "~ ~\N ~ O
~
Note: This sequence must be conducted under dry conditions with dry solvents other~,vise
epimerisation will occur.
Methyl iodide ( 1 Oml) was added to Boc-(D)-Met-(O-Ala-OMe (8g) in a mixture of DMF
(20ml) and dichloromethane (20ml) and the mixture was allowed to stand for 16 hours and
then evaporated to dryness. Further dichloromethane (2xSOml) was added and evaporated to
-
- ~ CA 02242809 l99X-07-09
WO 97131023 PCT/GB97100438
- 32 -
remove residual methyl iodide and the residue was dissolved in a mixture of DMF (300ml)
and dichlolol.,c~ n~ (300 ml). The mixture was cooled to ~5~C and sodium hydride (0.76g
of an 80% dispersion in mineral oil) was added in one portion and the ~ Ul~ was stirred at
this temperature for 2 hours. Saturated aqueous ~mmonium chloride (50 ml) was added and
5 the mixture was evaporated to dryness and then partitioned between water and ether. The
ether extract was washed with brine and dried and evaporated to give a gurn which was
purified by flash chromatography on a sinter funnel (25% ethyl acetate:hexane to 100%
ethylacetate) to give methyl (2O-2-[(3O-3-(~ butyloxycarbonyl]amino)-2-oxo-
pyrrolidin-l-yl]propionate as a gum (4.2g) which crystallised on st~n-ling NMR (CDCl3):
1.4 (s, 9H), 1.4 (d, 3H~, 1.8 (m, lH), 2.6 (m, lH), 3.4 (m, 2H), 3.7 (m, 3H), 4.2 (m, lH), 4.9
(~, lH), 5.2 (bs, IH).
1.3 Synthesis of (2~-2-l(3~)-3-(~-[9-fluorenylmethyloxycarbonyllamino)-2-oxo-
pyrrolidin-l-yllpropionic acid (Fmoc-TIb-OH)
--~ ~~N~ N J~--OH
0
Methyl (2O-2-[(3~)-3-(~-~tert-butyloxycarbonyl~amino)-2-oxo-pyrrolidin- 1 -yl]propionate
(4g) was refluxed in a mixture of acetone (60ml), water (40ml) and concentrated hydrochloric
acid (24ml) for 3 hours and then the mixture was evaporated to dryness. Water was added and
20 the evaporation repeated. The residue was dissolved in water ~ I Sml) and excess solid sodium
bicarbonate was added. 9-Fluorenylmethyl s~ cinimidyl carbonate (5.2g) in acetone (30ml)
was added. The mixture was stirred for 16 hours and then the solvent was removed by
evaporation and the residue was partitioned between water and ether. The aqueous layer was
separated, its pH adjusted to ~3 with hydrochloric acid, and extracted with dichloromethane.
25 -The organic layer was washed with water, dried (MgSO4) and evaporated to give a white foam
which crystallised on trituration with ether to give (2O-2-[(3O-3-~-[9-
fluorenylmethyloxycarbonyl]arnino)-2-oxo-pyrrolidin-1-yl]propionic acid ~4.2g) as a white
,
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solid, mp. 191-3~C (dec); NMR (CDCl3): 1.4 (d, 3H), 2.0 (m, lH), 2.6 (m, lH), 3.4 (m, 2H),
4.2 (t, lH), 4.4 (m, 3H), 4.9 (m, lH), 5.8 (bs, lH), 7.4 (m, 4H), 7.6 (d, 2H), 7.7 (d, 2H).
1.4 Synthesis of Phv-~la-Ala-Ala-Lys-Val-IIb-Ala-Pip-NH2 (SEQ ID NO: 1)
S
The peptide was prepared by Fmoc solid phase synthesis starting with Fmoc Rink Amide
MBHA Resin (Novabiochem, 0.50g, 0.25mmoles) in a Bond Elut tube (Varian, 1 Sml, fitted
with a filter in the bottom).
10 (a) The resin was deprotected using a 20% solution of piperidine in DMF (two treatments
with 5ml for 10 minllt~?~ each). After deprotection the resin was thoroughly washed with
DMF (S x 1 Oml).
(b) Acylation was carried out by addition of a solution of Fmoc-Pip-OH (353mg, lmmol),
DMF(l.Sml), HOBT (165mg, lmmol) and diisopropylcarbodiimide (155 microlitres, lmmol).
The coupling was left for approximately 30 minllt~s, washed with DMF (5xlOml) and a small
portion of the resin checked for completion of acylation using the Kaiser test (E. Kaiser~ et al,
(1970), Anal. Biochem. 34, S9S)
20 The above deprotection (a) and coupling cycle (b) were repeated using,
Fmoc-Ala-OH (311mg, lmmol);
Fmoc-IIb-OH (394mg, lmmol);
Fmoc-Val-OH (339mg, lmmol);
Fmoc-Lys(Boc)-OH (468mg, lmmol);
Fmoc-Ala-OH (311mg, lmmol);
Fmoc-Ala-OH (311mg, lmmol);
Fmoc-Ala-OH (311 mg, l mmol); and
S-Phenylvaleric Acid (178mg, lmmol);
30 In each case the coupling time was around 30 minutes and a small portion of the resin was
checked for completion of acylation by the Kaiser~test. The phenylvaleric acid required a
double couple to obtain a positive result by the Kaiser test.
CA 02242809 1998-07-09
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The peptide was cleaved from the resin using a mixture of trifluoroacetic acid (7.9ml) and
triethylsilane (0.395ml). After 2 hours the resin was washed with dichlorometh~n~
(approximately 150 ml) and the resulting solution evaporated to dryness. The res~llting solid
was partitioned between ether (25ml) and water (25ml) and then the ether was extracted with
further portions of water (2x25ml). The aqueous phases were combined and freeze dried~
The crude product was purified using pl~dldlive RP-HPI C (Vydac 218TP1022 column,
250mm x 22mm), loading the crude material in 5mls of 20% acetonitrile~water with 200
10 microlitres of DMF. Elution was carried out with a gradient of acetonitrile-water cont~ining
0.1%TFA (15-35% acetonikile) over 80 minutes at a flow rate of 10ml/minute. The fractions
cont~ining product were combined and freeze dried to give
Phv-Ala-Ala-Ala-Lys-Val-IIb-Ala-Pip-~H2 (SEQ ID NO: 1 ~ as a white solid (84mg; 35%
yield).
The product was characterised by HPLC, mass spectroscopy and amino acid analysis.
RP-HPLC Vydac C 18 column, 21 8TP54! 4.6x250mm. eluting with acetonitrile and water
cont~ining 0.1% TFA, using a 10-50% acetonitrile gradient over 30 minutes. flow rate 1.0
20 rnl/minute, indicated 100% purity, retention time 18.56 min~ltl~s: mass spectrometry, m/e
(positive electrospray (ES+)3 954.6 (MH ); arnino acid analysis (acid hydrolvsis over 24
hours using a solution of 6N HCI containing 1% phenol at 130~~) gave (Ala+IIb) 4.80. Val
l . I 1, Lys 1.07.
Fmoc-Pip-OI I was obtained by an analogous method to that described in E. Atherton
and R. ~. Sheppard ("Solid phase peptide synthesis: a practical approach", IRL press. 1989.
page 51) for~-Fmoc-L-methionine:
Fmoc-Pip-OH: NMR (DMSO-d6) 1.3 (m, 2H), 1.7 (m. 2H). 2.5 (m, 2H), 2.9 (t, 2H), 3.7 (m,
lH), 4.2 (t, lH), 4.4 (d, 2H), 7.4 (m, 4H), 7.7 (d, 2H), 7.9 (d, 2H); mass spectrometry m/e
30 ~ES+) 352.2 (MH )
.
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-3S-
Examl)le 2. Phv-Ala-Ala-Ala-Lys-Val-III-Ala-Pip-NH2 and separation of isomers.
(SEQ ID NO: 2)
2.1. Synthesis of (RS)-2-allyl-N-(benzyloxycarbollyl~proline
N ~rOH
0~o ~
N-benzyloxycarbonylproline methyl ester (13g) in THF (20ml) was added dropwise to lithium
diisopropylamide (27.5 ml, 2M in hexane/THF) in THF (lOOml) at -78~C under nitrogen. The
mixture was stirred for 3~) minllt~c and then allyl iodide (5.5ml~ was added dropwise and the
mixture stirred for a further 30 minutes and then allowed to warm to ambient te,.ll~e,ature.
10 The mixture was then added to aqueous ammonium chloride (200ml) and extracted with ether
(2x200ml). The ether layer was evaporated and the residue was purified by chromatography
on silica using a gradient of hexane increasing to 20% ethyl acetate:hexane. The appropriate
fractions, evaporated to dryness, gave methyl (~)-2-allyl-N-(benzyloxycarbonyl)prolinate
(9g) as an oil.
8.5 g of this material was dissolved in m~th~n~l (40ml) and sodium hydroxide (4.5g) added in
water (20ml) and the mixture refluxed for 60 minl~t~ The pH of the mixture was then
adjusted to 7 with concentrated hydrochloric acid and the methanol was removed by
evaporation. The pH of the mixture was adjusted to 3 and the mixture was extracted with ether
20 (2xSO ml) . The combined ether extracts were evaporated to give (~)-2-allyl-N-
(benzyloxycarbonyl)proline as a gum: NMR (d6-DMSO (373K)): 1.9 (m, 2H), 2.1 (m, 2H),
2.6 ~q, lH), 2.9 (q, lH), 3.4 (m, lH), 3.6 (m, lH), 5.0 (m, 4H), 5.75 (m, IH), 7.3 (m, SH).
2.2 Synthesis of ~ 2-allyl-N-(benzyloxycarbonyl)prolyll-(O-alanine methyl ester
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o
HOBT (7.7g), N-methylmorpholine (6.6g3, L-alanine methyl ester hydrochloride (4.5g) and 1-
~3-dimethylaminopropyl)-3-ethyl-carbodiimide (5.7g) was added to (~)-2-allyl-N-
~benyloxycarbonyl~proline (6.5g) in DMF (30ml) and the mixture was stirred for 18 hours
5 and then evaporated. The residue was partitioned between ether and water, filtered to remove
HOBt and the organic layer separated. The organic layer was evaporated and the residue was
purified by chromatography on silica using a gradient of 20% ethyl acetate in hexane
illeleasillg to 50% ethyl acetate in hexane. The a~ iate fractions were combined and
evaporated to dryness to give [(~)-2-allyl-N-(benzyloxycarbonyl)prolyl~-(O-alanine methyl
10 ester (7g); NMR (db-DMSO (373K)): some doubling of peaks due to the mixture of
diastereoisomers, 1.25 and 1.3 (2d, 3H), 1.75 (m, 2H~, 2.2 (m, 2H), 2.65 (m, lH), 2.9 (m, IH),
3.4 (m, lH), 3.65 (2s, 3H), 3.7 (m, IH), 4.3 (2q, lH), 5.0 (m, 4H), 5.7 (m, IH), 7.3 (m, SH),
7.4 and 7.5 (bs, lH).
2.3 SynlLesis of m~ethyl (0-2-(1-benzyloxycarbonyl-6-oxo-1,7-diazaspiro~4.41non-7-
yl)propionate. (Cb~III-OMe)
~ . O
o~o
Osmium tetroxide ( 1 .Sml of 4% aqueous solution) was added to ~ 2-allyl-N-
~benzyl~ycall,onyl)prolyl]-(S)-alanine methyl ester (I .45g) in a mixture of methanol (30ml)
and water (20ml). The mixture was stirred under argon for 10 min~1t~s and then sodium
periodate (2.45g) was added in portions: The mixture was stirred for 2 hours and then water
~lOOml) was added and the mixture was extracted with ethyl acetate (2x70ml). The combined
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extracts were dried and evaporated to give 1.4g of a gum. The gum was dissolved in
dichloromethane (30ml) and triethylsilane (0.65g) and then trifluoroacetic acid (4g) were
added dropwise. The mixture was stirred for 3 hours, evaporated and the residue partitioned
between aqueous sodium bicarbonate and ether. The ether extract was separated and
S evaporated to dryness. The residue was purified by chromatography on silica using a gradient
of 25% ethyl acetate in hexane increasing to 100% ethyl acetate. The a~ iate fractions
were combined and evaporated to dryness to give methyl (~-2~ benzvloxycarbonyl-6-oxo-
1,7-diazaspiro[4.4]non-7-yl)propionate (0.8g); NMR (d6-DMSO (373K)): some doubling of
peaks due to the mixture of diastereoisomers, 1.25 and 1.35 (2d, 3H), 1.95 (m, 6H), 3.1-3.5
(m, 4H). 3.6 and 3.65 (2s, 3H), 4.5 and 4.65 (2q, lH), 5.05 ~m, 2H)~ 7.25 (m, SH).
2.4 Synthesis of (~-2-(6-oxo-1,7-diazaspirol4.4]non-7-yl)propionic acid (H-III-OH)
Pn~s~ carbonate (2.5 g) was added to methyl (0-2-(1-benzyloxycarbonyl-6-oxo-1,7-
diazaspiro[4.4]non-7-yl)propionate (3.3g) in a mixture of methanol (40ml) and water (40ml)
and the mi~Lu,c was stirred at ambient tt;lll~cldLulc for 10 hours. The pH was adjusted to ~5
with concentrated hydrochloric acid and the mixture was evaporated to dryness. The residue
was dissolved in water (40ml) and the pH adjusted to 3 with concentrated hydrochioric acid.
The mixture was then extracted with dichloromethane (2xSOml). The combined extracts were
20 dried ( MgSO4) and evaporated to yield a foam (2.8g). The foam was dissolved in methanol
(20ml) and cyclohexene (0.7g) was added followed by 10% PdlC (O.Sg). The mixture was
refluxed for 2 hours, cooled, filtered and the filtrate was evaporated to give (0-2-(6-oxo- 1,7-
di~as~ o[4.43non-7-yl)propionic acid as a foam (1.9g); NMR (d6-DMSO): some doubling of
peaks due to the mixture of diastereoisomers, 1.25 and 1.3 (2s. 3H), 1.8 (m, 4H), 2.0 (m, 2H),
3.0 (m, 2H), 3.3 (m, 2H), 4.5 (m, IH).
2.5 Synthesis of (0-2-[1-(9-fluorenylmethyloxycarbonyl)-6-oxo-1,7-diazaspiro-
[4.41non-7-yllpropionic acid ~Fmoc-III-OH)
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- 38 -
OH
0~ 0
o
Excess solid sodiurn bicarbonate was added to (0-2-(6-oxo-1,7-dia~ o[4.4]non-7-
yl)propionic acid (0.42g) in water (2ml) and then 9-fluorenylmethyl ~ çinimidyl carbonate
(0.7~) in acetone (3ml) was added. The mixture was stirred for 18 hours. The mixture was
S then added to water ( 1 Oml~, extracted with ether ( 1 Oml) and the aqueous layer separated. (The
ether extracts were discarded). The pH of the aqueous layer was adjusted to ~3 with
concellLldled hydrochloric acid and then it was extracted ~,vith dichloromethane (2xlOml). The
combined extracts were separated, dried (MgSO4) and evaporated to give (0-2-~1-(9-
~luorenylmethyloxycarbonyl)-6-oxo-1,7-diazaspiro[4.4]non-7-yl~propionic acid (0.62g) as a
10 white foam; NMR ~d6-DMSO (373K)): some doubling of peaks due to the mixture of
diastereoisomers, 1.3 ~2d, 3H), 1.6-2.0 (m, 6 H), 3.05 (m, IH), 3.2-3.45 (m, 3 H), 4.2-4.4 (m,
l H), 4.5 ( m, 1 H), 6.2 (s, 2H), 7.35 (m, 4H), 7.8 (m, 4H).
2.6 Synthesis of Phv-AIa-AIa-A1a-L ys-V al-III-Ala-Pip-N H2 a~ d separation of
isonners. (S E Q ID N O: 2)
The first part of the synthesis was performed using similar methodology to that described for
Example 1.4, but using Fmoc-III-OH in place of Fmoc-IIb-OH. In this case the acylations
were performed by activation of the acids in the following way: Acid ( I mmol), HBTU ( I
mmol), diisopropylethylamine (2 rnmol), DM~ (4ml). All couplings were seen to be20 complete by the Kaiser test after less than 45 minl~t~ This methodology was used to give
Fmoc-Val-III-Ala-Pip-NH-Resin. The peptide resin was then transferred to an ABI 431
automated peptide synthesiser where the rem~ining residues in the sequence were coupled
following the rn~nllf~ctllrer~s recomm~nc~ed conditions for single acylations incorporating
HBTU / HOBT chemistry, as follows. After deprotection, the resin was washed with DME;
(10 x 10-20 ml). The carboxylic acid (lmmol) was activated with ~BTU ( I equivalent).
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HOBT (1 equivalent) and DIPEA (2 equivalents) in DMF for approximately 11 ~ s
before transfer to the resin. The acylation was carried out for approximately 60 minntt~ and
then the resin was washed with DMF ( 10 x 10-20 ml). Fmoc deprotection at each stage was
carried out using a 20% solution of piperidine in DMF (two treatments with 5 ml for 10
S min~lt~c each). ~fter each deprotection, the resin was thoroughly washed with DMF ( 5 x 10
ml).
The peptide was cleaved from the resin by treatment with a mixture of trifluoroacetic acid
(TFA), triethylsilane (TES) and water (lOml, 90:5:5) for 1.5 hours. The resin was filtered off,
washed thoroughly with TFA~ the collected filtrate concentrated to dryness by rotary
l 0 evaporation and then the resulting residue triturated with ether to yield Phv-Ala-Ala-Ala-Lys-
Val-III-Ala-Pip-NH2 (SEQ ID NO: 2) cont~;ning two major components (RP-HPLC (Vydac
218TP54 C18 column) indicated a 52:48 mixture). The more polar diastereomer eluted with a
retention time of 12.5 minnt~ and the less polar diastereomer with a retention time of 16.5
minlltcS using an acetonitrile:water (con~ining 0. I% TFA) gradient of 20-50% over 20
minutes at a flow rate of lml/minute. The peptide was further purified by ple~al~tive RP-
HPLC, essentially as described for Example 1.4, to give the two isolated components, both
>95% pure by RP-HPLC. The more polar diastereomer was more potent than the less polar
diastereomer in Tests A and B and was characterised as follows:
RP-HPLC (20-50% acetonitrile gradient over 20 minllt~s7 flow rate 1 ml/minute) retention
time = 12.3 minutes; mass spectrometry, m/e (ES+) 994.5 (MH+); amino acid analvsis gave
Ala 3.99, Lys 1.20, Val 0.73.
Examl~le 3. Phv-Ala-Lys-Ala-IIb-Ala-Ala-Ala-Pip-NH2 (SEQ ID NO: 3)
The synthesis was performed m~nll~lly, and the purification carried out. using similar
methodology to that described for Example 1.4, coupling and deprotecting the Fmoc-
protected amino acids and Fmoc-Ilb-OH in the apl-lu~Jliate order. The product was
characterised by HPLC, mass spectroscopy and amino acid analysis in a similar manner to
~ that described for Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~ining
0.1% 7'FA, using a 25-40% acetonitrile gradient over 20 minntes, flow rate 1 .Oml/minute~
retention time = 5.56 minutes; mass spectrometry, m/e (ES+) 926.5 (MH~): amino acid
analysis gave ~Ala+lIb) 5.87, Lys 1.12.
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Examl~le 4. Phv-l[le-Ala-Ala-Arg-Thr-IIb-Ala-Papa-NH2 (SEQ ID NO: 4)
The synthesis was performed m~ml~liy, and the purification carried out, using similar
methodology to that described for Exarnple 1.4, coupling and deprotecting the Fmoc-Papa-
OH ~used instead of Fmoc-Pip-OH), Fmoc-protected amino acids and Fmoc-IIb-OH in the
a~pl~pliate order. The product was characterised by HPLC, mass spectroscopy and amino
acid analysis in a similar manner to that described for Exarnple 1.4, RP-HPLC (e~uting with
acetonitrile and water cC~ntzlinin~ 0. I % TFA, using a 20-50% aceto litrile gradient over 30
min~ltec, flow rate 1 .Oml/minute) retention time = 1 7.08minutes; mass spectrometry, m/e
~ES+) 1048.7 (MH~); amino acid analysis gave Arg 0.98, Ala 3.00, IIb 1.27, Thr 0.86, Ile
O.g8.
Fmoc-Papa-OH was obtained by an analogous method to that described in E. Atherton
and 1~. C. Sheppard ("Solid phase peptide synthesis: a practical approach", IRL press, 1989,
page S I ) for lV-Fmoc-L-methionine:
Fmoc-Papa-OH: NMR (DMSO-d6) 3.5 (s, 2H), 4.25 (t~ lH), 4.5 (d, 2H), 7.1 (d, 2H), 7 4 (m,
6H), 7.75 (d, 2H), 7.9 (d, 2H), 9.6 (s, lH); mass spectrometry m/e (ES ) ~72.1 (M-H)-
Example 5. Phv-Arg-Ala-Ala-Ala-Val-IIb-Ala-Pip-NH2 (SEQ ID NO: S)
The synthesis was p. ~ ed m~n-l~l1y using similar methodology to that described for
Example 1.4, coupling and deprotecting the Fmoc-protected amino acids and Fmoc-IIb-OH in
the a~lulJliate order. The product was purified usin~, preparative RP-HPLC in a similar
manner tû that described for Example 1.4, eluting with a gradient of acetonitrile-water
cont~inin~; 0.1%TFA (15-40% acetonitrile) over 60 minutec at a flow rate of 10ml/minute.
The product was ch~r;~rt~rised by HPLC, mass spectroscopy and arnino acid analysis in a
s,imilar manner to that described for Example 1.4; RP-HPLC (eluting with acetonitrile and
water Cont~inine 0.1% TFA, using a 10-50% acetonitrile gradient over 30 minutes, flow rate
1 0ml/minute) retention time--18.28 minutlos; mass spectrometry, m/e (ES+) 982.5 (MH );
~mino acid analysis gave Arg 1.12, (Ala + IIb) 3.8, Val 1.12.
ExamPle6. Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NH2 (SEQ ID NO: 6)
The first part of the synthesis was performed manually, using similar methodology to that
described for Example 1.4, coupling and deprotecting the Fmûc-Papa-OH (used instead of
Fmûc-Pip-OH), Fmoc-protected amino acids and Fmoc-Ilb-OH in the appropriate order to
CA 02242809 1998-07-09
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-41-
give Fmoc-Thr(OBu)-IIb-Ala-Papa-NH-resin. The peptide resin was then transferred to an
tc-m~tf d synthssi~er (ACT 357) and the rem~inin~ residues were coupled following the
m~n~lf~ct-lrer's recommt-nAecl conditions for single acylations incorporating HBTU/HOBT
ch~mi~try. The peptide was cleaved from the resin and purified by ~ ~dLive RP-HPLC
S using a similar procedure to that described in Example 1.4. The peptide was characterised by
HPLC, mass spectroscopy and amino acid analysis in a similar manner to that described for
FY~mrle 1.4; RP-HPLC (eluting with acetonitrile and water cont~inin~ 0.1% TFA, using a
10-50% acetonitrile gradient over 30 minlltPc, flow rate 1.0ml/minute) retention time =
18.21min-1t~s; mass spectrometry. m/e (ES+) 1006.4 (MH ); amino acid analysis gave Arg
1.10, Ala 3.88, IIb 1.04, Thr 0.95.
Example 7. Phv-Ala-Ala-Ala-Arg-Val-IIb-Ala-Papa-NH2 (SEQ ID NO: 7)
The first part of the synthesis was p~,.ru~llled m~nn~ql Iy, using similar methodology to that
described for Example 1.4, coupling and deprotecting the Fmoc-Papa-OH (used instead of
15 Fmoc-Pip-OH), Fmoc-protected amino acids and Fmoc-IIb-OH in the ~p~ iate order to
give Fmoc-Val-IIb-Ala-Papa-NH-resin. The peptide resin was then transferred to an
automated syntht ci~-Dr (ABI 431) and the renns~ining residues coupled following the
m~nl-f;~n~lrer's recommended conditions for single acylations incorporating HBTU/HOBT
chemistry. The peptide was cleaved from the resin and purified by p~c;~ Li~e RP-HPLC
20 using a similar procedure to that described in Example 2.6. The peptide was characterised by
HPLC, mass spectroscopy and amino acid analysis in a similar manner to that described for
Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~ininE 0.1% TFA, using a
l~-so% s~cetQnitrile gradient over 30 minnt~s, flow rate 1.0ml/minute) retention time = 20.87
...i....l~, mass spectrometry, m/e (ES+) 1004.6 (MH ); amino acid analysis gave Arg 0.96,
(Ala + Ilb) 5.05, Val 0.97.
Esample 8. Phv-Ala-Arg-Ala-Arg-Val-~Ib-Gly-Papa-NH2 (SEQ ID NO: 8)
The first part of the synthesis was performed m~nn~lly, using similar methodology to that
described for Example 1.4, coupling and deprotecting Fmoc-Papa-OH (used instead of Fmoc-
30 Pip-O~, the Fmoc-protected amino acids and Fmoc-IIb-OH, to give Fmoc-Val-lIb-Gly-Papa-
NH-resin. The peptide resin was then transferred to an automated synthesiser (ABl 431~ and
the r~mz~ining residues coupled following the m~nnf~ct-lrer's recommended conditions t'or
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single acylations incorporating HBTUtHOBT ch~ try, The peptide was cleaved from the
resin and purified by ~p~dLi~e RP-HPLC using a similar procedure to that described in
Exarnple 2.6. The peptide was char~tPri.ced by HPLC, mass spectroscopy and arnino acid
analysis in a similar manner to that described for Example 1.4; RP-HPLC (eluting ~,vith
~ acetonitrile and water cont~ining 0.1% TFA, using a 10-50% acetonitrile gradient over 30
minllt~ flow rate l.Oml/minute) retention time = 18.12 minnt~-~; mass spectrometry, m/e
(ES+) 538.4 (M+2H ~; amino acid analysis gave Arg 0.92, Ala 2.16. (Gly + IIb~ 2.12, Val
0.88.
10 Example 9. Phv-Ala-lle-Ala-Arg-Val-IIb-Ala-Pip-NH2 (SEQ ID NO: 9)
The synthesis was ~clrs,ll.led m~nl-~lly, and the purification carried out, using similar
methodology to that described for Example 1.4, coupling and deprotecting the Fmoc-
protected amino acids and Fmoc-IIb-OH in the al""o~liate order. The product was
characterised by HPLC, mass spectroscopy and arnino acid analysis in a similar manner to
15 that described for Example 1.4; RP-HPLC (eluting with acetonitrile and water Cont~ining
0.1% TFA, using a 10-50% acetonitrile gradient over 30 min~ltes flow rate 1.0ml/minute)
retention time = 23.4 minutes; mass spectrometry, mte (ES~) 1024.6 (MH ); amino acid
analysis gave Arg I.15, (Ala + I~b) 3.88, Val 0.96, ~le 1.02.
20 Example 10. Phv-Ala-Arg-Ala-His-Val-IIb-Ala-Papa-NH2 (SEQ ID NO: 10)
The first part of the synthesis was performed manually, using similar methodology to that
described for Example 1.4, coupling and deprotecting the Fmoc-Papa-OH (used instead of
Fmoc-Pip-OH), Fmoc-protected arnino acids and Fmoc-IIb-OH in the appropriate order to
give Fmoc-Val-lIb-Ala-Papa-NH-resin. The peptide resin was then Lldll~f~cd to an2~ automated synthesiser (ABI 431) and the r~m~ining residues coupled following the
m~nllf~rturer's recornmended conditions for single acylations incorporating HBTU/HOBT
chemistry. The peptide was cleaved from the resin and purified by ple~alnlive RP-HPLC
using a similar procedure to that described in Example 2.6. The peptide was characterised by
HPLC mass spectroscopy and arnino acid analysis in a similar manner to that described for
Exarnple 1.4; RP-HPLC (eluting with acetonitrile and water cont~iningO.1% TFA, using a
20-50% acetonitrile gradient over 30 minllt~, flow rate 1.0ml/minute) retention time =
CA 02242809 1998-07-09
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18.8minl~tes; mass spectrometry, m/e (ES~) 1070 6 (MH+); amino acid analysis gave Arg
1.02, (Ala + IIb) 4.12, Val 0.90, His 0.95.
Example ll. Phv-Ala-Ala-Asn-Arg-Val-IIb-Ala-Pip-'NH2 (SEQ ID NO: 11)
S The first part of the synthesis was ~t;,rol,lled m~nll~lly, using similar methodology to that
described for Example 1.4, coupling and deprotecting the Fmoc-protected arnino acids and
Fmoc-Ilb-OH in the ~ p,;ate order, to give Fmoc-lIb-Ala-Pip-NH-resin. The peptide resin
was then L~ rGIlGd to an automated synth~ci~r (ABI 431 ) and the rern~ining residues
coupled following the m~n1lf~turer's recommentled conditions for single acylations
10 incorporating HBTU/HOBT chçmi~try. The peptide was cleaved from the resin and purified
by ~Ic~aldlive RP-HPLC using a similar procedure to that described in Exarnple 2.6. The
peptide was ch~r~terised by HPLC, mass spectroscopy and amino acid analysis in a similar
manner to that described for Example 1.4; RP-HPLC (eluting with acetonitrile and water
cont~inin~ 0.1% TFA, using a 10-50% acetonitrile gradient o~er 30 minnt~c. flow rate
1.0ml/minute) retention time = 17.66 min11te~; mass spectrometry, m/e (ES+) 1025.5 (MH );
arnino acid analysis gave Arg 1.04, Ala 2.94, Val 0.95~ Asp 1.05.
Example 12. Phv-Ala-Arg-Ala-IIb-AIa-Ala-Ala-Pip-~H2 ~SEQ ID NO: 12)
The first part of the synthesis was carried out automatically on an ABI 431 automated
20 synthe~ r using similar methodology to that described in Example 2.6~ coupling the Fmoc-
protected arnino acids in the appropriate order, to give Fmoc-Ala-Ala-Pip-NH-Resin. The
rçm~in<lcr ofthe synthesis was p~ ed m~nll~lly, and the product purified by lvlG~dlive
RP-HPLC, using a similar procedure to that described in Example 1.4. The peptide was
characterised by HPLC, mass spectroscopy and amino acid analysis in a similar manner to
25 that described for Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~inin~
0.1% TFA. using a 10-50% acetonitrile ~r~-lient over 30 minmec~ flow rate 1.0ml/minute)
retention time = 16.68 min~ltP~; mass ~ue~;Llollletry~ m/e (ES+) 954.6 (MH ); amino acid
- analysis gave Arg 1.09, (Ala + IIb) 5.88.
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-44-
Examplel3. Phv-Ala-Arg-Ala-Ala-Thr-IIb-Gly-N ~(C H~)3N H.C(=N H).N H2
(SEQ ID NO: 14)
(a~ Preparation of Phv-Ala-Arg-Ala-Ala-Thr-IIb-Gly-NH~CH2)3NH2
(SEQ ID NO: 13)
2-Chlo~ iLyl chloride resin, (0.4g ca. 0.25 mmole of available chlorine) was converted to (3-
aminopropyl)arnino-resin by reaction witn 1,3-diaminopropane (0.42 ml) in DMF (4ml) for I
hour at ambient t~ eldLule. After thorough washing with DMF, the resin was transferred to
an Applied Biosystems 430A peptide synthesiser and the sequential coupling and deprotection
10 ofthe a~ro~ le protected amino acids was carried out following the msmllf~cturer's
recommended conditions. The resin was then washed with meth~n<-l and dried (0 722 g). The
peptide was cleaved from the resin and purified by ~le~ dli~e RP-HPLC using a similar
procedure to that described in Example 2.6. The product was characterised by HPLC. mass
spectroscopy and amino acid analysis in a similar manner to that c}escribed for Example 1.4;
15 RP-HPLC (eluting with acetonitrile and water cont~ining 0.1% TFA, using a 10-50%
act;lo~ ile ~rat~ nt over 40 minllt~c, flow rate l.2 ml/minute) retention time = 17.76 minllt~c
There was thus obtained Phv-Ala-Arg-Ala-Ala-Thr-IIb-Gly-NH(CH2)3NH2 (SEQ ID NO: 13)
(329 mg after freeze drying); mass ~,~e~ unletry, m/e (ES+) 916.5 (MH ).
~b) Preparation of Phv-Ala-Arg-Ala-Ala-Thr-lIb-Gly-NH(CH2)3NH.C~=NH).NH2
(SEQ ID NO: 14)
Phv-Ala-Arg-Ala-Ala-Thr-rIb-Gly-NH(CH2)3NH2 (SEQ ID NO: 13) (329 mg) was dissolved
in a ~ u-c of water (5 ml) and acetonitrile (5 ml) and 1-H-pyrazole-l-carbox~ iin~
hydrochloride (132 mg, 3 equivalents) and diisopropylethylarnine (52 microlitres, I
equivalent) were added. The mixture was stirred at ambient ~ eL~Lure for 4 days. The
mixture was purified by ~ ald~ive ~P-HPLC and characterised by HPI,C, mass
spectroscopy and amino acid analysis in a similar manner to that described for Example 1.4.
There was thus obtained
Phv-Ala-Arg-Ala-Ala-Thr-IIb-Gly-NH(CH2)3NH.(~(=NH).NH2 (SEQ ID NO: l4) (178 mg);RP-HPL~ (eluting with acetonitrile and water cont~ining 0.1% TFA, using a 10-50%acetonitrile gradient over 40 minutes~ flow rate 1.2 ml/minute) retention time = 18 4~
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-45-
mimltes; mass spectrometry, m/e (ES+) 958.5 (MH+); amino acid analysis gave Thr 0.86, Gly
0.90; Ala 3.20, Arg 1.00
Examnle 14. Phv-Ala-Arg-Ala-Arg-Thr-IIb-Gly-NHEt (SEQ ID NO: 15)
The first part of the synthesis was carried out on an Applied Biosystems 430A synth~si~er and
the sequential coupling and deprotection of the d~ro~l;ate protected amino acids was carried
out following the m~nL f~rturer's recommended conditions, starting from Boc-Gly-O-Benzyl
ester resin (Merrifield resin) ((~,54g, 0.25 mmole). The resin was washed with methanol and
dried (871mg) and then stirred gently with DMF (lOml) and 2M ethylarnine in methanol (10
10 ml) at ambient temperature for 7 days. RP-HPLC indicated a rapid release of methyl ester
into solution followed by a slow conversion to the slightly more hydrophilic N-ethylamide.
The mixture was filtered and the resin was washed with DMF. Evaporation of the solvents
afforded the crude, protected peptide as the N-ethylamide. The peptide was deprotected using
a mixture of TFA, TES and water (90: 5: 5) and purified by l,lepa.dlive RP-HPLC using a
15 similar procedure to that described in Example 2.6 to give Phv-Ala-Arg-Ala-Arg-Thr-IIb-Gly-
NHEt (SEQ ID NO: 15) (168 mg). The peptide was ch~r~rt~rised by HPLC, mass
spectroscopy and amino acid analysis in a similar marmer to that described for Example 1.4;
RP-HPLC (eluting with acetonitrile and water co,.~ 0.1% TFA, using a 10-50%
acetonitrile gr~-lient over 40 min, flow rate 1.2 ml/minute) retention time = 17.85 m;nlltec;
20 mass spectroscopy, m/e (ES+) 972.5 (MH+); amino acid analysis gave Thr 0.83, Gly 1.03, Ala
2.17, IIb û.9, Arg 2.00.
Examl)les 15 and 16.
Phv~ ap-Ala-Ala-Ala-Thr-lIb-Ala-Papa-NH2 (Example 15) (SE~2 ID NO: 16)
Phv-Gap(Me~4-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NH2 (Example 16) (SEQ ID NO: 17)
(a) P~ ~d~ionofPhv-Dap-Ala-Ala-Ala-Thr-Ilb-Ala-Papa-NH2 (SEQ ID NO: 36)
The synthesis was performed automatically on an Applied Biosystems 430A synthesiser,
30 using Fmoc Rink Amide resin ~0.25 mmole), and the sequential coupling and deprotection of
the appropriate protected amino acids was carried out following the manufacturer's
recommended conditions. The Dap residue was incorporated by using Fmoc-Dap(Boc)-OH at
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W 097/31023 PCT/GB97/00438
_~6-
the a~plupliate stage in the synthesis. The completed resin was dried. Yield 1.292 g (weight
gain, 792 mg). The peptide was cleaved from the resin and purified by p.Gpa dLive RP-HPLC
using a similar procedure to that described in Exarnple 1.4, but using a Dynamax 60A column
(one inch internal diameter) eluting with acetonitrile and water cnnt~inin~ 0.1% TFA
(gradient 10-40% acetolliLl;le) over 60 minl-tes at a flow rate of 12 ml/minute, to give Phv-
Dap-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NH2 (SEQ ID NO: 36) (304 mg). The peptide was
characterised by HPLC and mass spectroscopy in a similar manner to that described for
Exarnple 1.4; RP-HPLC (eluting with acetonitrile and water cont~inin~ 0.1% TFA, using a
10-40% acetonitrile gradient over 40 m~l, flow rate 1.2 ml/minute) retention time = 24.5
10 ~ e~:, mass spectroscopy, m/e (ES+~ 937.1 (MH+).
(b) The Dap peptide from (a) was dissolved in water (7 ml) and acetonitrile (~ml~ and
treated with 1-H-pyrazole-1-carboxyamidine hydrochloride (130 mg) and diisopropylethyl
amine (53 microlitres3 at ambient temperature for 1 week. RP-HPLC indicated only 40%
15 conversion to the corresponding Gap peptide. Attempted p~ ,alc~Live RP-HPLC failed to
completely separate the two compounds. Accordingly, the rnixture was treated with HBTU
(87 mg) and diisopropylethylamine (50 microlitres) in DMF (50 ml) for 16 hours to convert
the unchanged Dap peptide to the tetrarnethylguanidine (GapMe4) peptide. The extra
hydrophobicity of this compound enabled an easier ~ alaLive HPLC (conditions as
20 described in (a)), which was carried out using identical condi~ions to those described in (a)
above, to give both
(i) Phv-Gap-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NH~ (SEQ ID NO: 16) (80mg) which was
characterised by HPLC, mass spectroscopy, and amino acid analysis in a similar manner to
that described in Example 1.4; RP-HPLC (10-40% acetonitrile gradient over 40 min, flow rate
1.2 mllminute) retention time = 26.2 minlltes; mass spectroscopy, rn/e (ES ~ 978.5 (MH~);
amino acid analysis gave Thr 0.90, Ala 4.05, IIb present, peak in Trp position for Gap.
and
(ii) Phv-Gap(Me)4-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NH2 (122 mg) (SEQ ID NO: 17), which
was characterised as for (i) above; RP-HPLC (eluant and gradient as in (i) above) retention
time = 28.4 minntec; mass spectroscopy, m/e (ES ) 1034.6 ~MH ), amino acid analysis gave
Thr 0.90, Ala 4.1 1. IIb present, peak in Trp position for Gap(Me)4.
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ExamPlel7. Phv-Arg-Ala-Ala-Ala-Thr-~Ib-Gly-NHCH2CH2 Morpholine
~SEQ ID NO: 18)
A protected fragment strategy was adopted. l~moc-Gly-O-Chlorotrityl resin (1mrnol, lg) was
plel)al~,d autom~tif~s31ly on an Applied Biosystems 430A synthesi~er by coupling rmoc-Gly-
5 OH (2mmol) with 2-chlorotritylchloride resin (lg, lmmol) in DMF (lOml) in the presence of
N-diisopropylethylamine (4mmol) and elongated by a double coupling strategy (in a similar
manner to that described in Exarnple 2.6) to give the protected peptide resin (2.15g). After
cleavage from the resin with dichloromcth,.n~- (30 ml), acetic acid (Sml) and trifluoroethanol
(Sml) at ambient temperature for 1 hour, the resin was filtered off and washed with
10 dichloromethane and acetic acid. Evaporation of the solvents and subsequent freeze-drying
from aqueous acetonikile afforded the required protected peptide acid (1.23 g). The peptide
was characterised in a similar manner to that described in Example 1.4; RP-HPLC (20-80%
acetonitrile gradient over 40 minlltes~ flow rate 1.2 ml/minute) retention time = 26.2 minut~c
The protected acid (235 mg, 0.2 mmol) was coupled to 4-(2-aminoethyl)morpholine (27
microlitres, 1 equivalent) in DMF (1 Oml3 with HBTU (76 mg, 1 equivalent3 and
diisopropylethylamine (70 microlitres3. Evaporation of tne solvent, deprotection with 90%
TFA/H20 and purification, using a similar procedure to that described in Example 1.4,
afforded the pure arnide (150 mg). The peptide was characterised by HPLC, mass
spectroscopy and arnino acid analysis in a similar manner to that described for Exarnple 1.4;
RP-~PLC (10-40% acetonitrile gradient over 40 minutec, flow 1.2 ml/minute~ retention time
= 18.6 minlltes; mass spectroscopy, m/e (ES ) 972.8 (MH ); arnino acid analysis gave Thr
0.6, Gly 1.02, Ala 2.94, IIb 0.9, Arg 1.00.
ExamPle 18. Phv-Arg-Ala-Ala-Ala-Thr-lIb-Gly-N(CH2CH2)2NCH2CH20-C~H2CH20H
(SEQ ID NO: 19)
Using an analogous procedure to that described in Example 17, but using 4-[2-(2-hydroxyethoxy)ethyl]piperazine in place of 4-(2-aminoethyl)morpholine, there was thus
obtained Phv-Arg-Ala-Ala-Ala-Thr-IIb-Gly-N(CH2CH2)2NCH2CH20-CH2CH20H (SEQ ID
NO: 19). The peptide was characterised by HPLC, mass spectroscopy and amino acid analysis
in a similar manner to that described for Example 1.4; RP-HPLC (using an eluant and gradient
CA 02242809 l99X-07-09
WO 97131023 PCT/GB97/00438
- 48 -
as used in Exarnple 17) retention time = 20.5 ~ Il/Ps; mass spectroscopy, m/e (ES ) 1016.8
(MH ); amino acid analysis gave Thr 0.89, Gly 1.02, Ala 2.98, IIb 1.05, Arg 1.00.
Examl~le 19. Phv-~rg-Ala-Ala-Ala-Thr-IIb-Gly-Pape-NHC(=NEI)NH2 (SEQ ID NO:
5 20)
Using an analogous procedure to that described in E~xample 17, but using 4-(2-
guanidinoethyl)aniline in place of 4-(2-arninoethyl3morpholine, there was thus
obtained Phv-Arg-Ala-Ala-Ala-Thr-IIb-Gly-Pape-NHC(=NH)NH2 (SEQ ID NO: 20). The
peptide was purified by ~ Li~te HPLC in a similar manner to that described for Example
1.4, but using a Vydac 201HS1022 column (one inch internal diameter) and eluting with a
gradient of acetonitrile-water cont~ining 0.1% TFA (10-35% acetonitrile) over 60 minllt~s at a
flow rate of 12ml/minute. The peptide was characterised by HPLC~ mass spectroscopy and
amino acid analysis; RP-HPLC (using an eluant and gradient as used in Exarnple 17) retention
time = 24.2 minllt~s; mass spectroscopy, m/e (ES~) 1021.1 (MH+); Amino acid analysis gave
Thr 0.8, Gly 0.95, Ala 2.86, Arg 1.00, IIb present.
4-(2-GIl~ni~inoethyl)aniline dihydrochloride was prepared as follows.
4-Nitrophenethylarnine hydrochloride ( 1.01 3g, Smrnol), 1 H-pyrazole- 1 -carboxarnidine
hydrochloride (0.733g, 5mmol) and N-diisopropylethylamine (0.88ml. ~mmol) in acetonitrile
20 -(lOml) and water (lOml) were stirred at arnbient tempGld~ulG for 16 hours. PlGp~d~ e RP-
HPLC was carried out in two portions using a Dynamax 60A, C18 column (one inch internal
diameter) eluting with acetonitrile and water c~mt~ining 0.1% TFA to afford pure 4-
nitroph.onf?thylgl-~ni~in~ The product was dissolved in water (lOOml) and one drop of
conce~ dl~d hydrochloric acid was added, followed by 5% palladiurn on carbon (lOOmg) and
25 hydrogen was bubbled through the solution for 4 hours. The catalyst was removed by
filtration and washed with water. Volatile m~tPri~l was removed by evaporation to give 4-(2-
gll~ni~lin~ethyl)aniline dihydrochloride as a brown foam (0.984 g) (after drying over
potassiurn hydroxide pellets).
Example 20. Phv-Ala-Ala-Ala-Arg-Thr-IIb-Ala-Papa-NH2 (SEQ ID NO: 21)
The first part of the synthesis was performed m~n~ lly, using similar methodology to that
described for Exarnple 2.6, using HBTU/DIPEA activation and coupling and deprotecting the
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W O 97/31023 PCT/GB97/OQ438 -49-
Fmoc-protected amino acids and Fmoc-IIb-OH in the ~.ol,.;ate order, to give Fmoc-IIb-Ala-
Papa-NH-resin. The peptide resin was then tr~n~f~rred to an automated synthesiser ~ABI 431 )
and the rem~ining residues coupled following the m~nuf~ch-rer's recommended conditions for
single acylations incorporating HBTU/HOBT çhemi~try. The peptide was cleaved from the
S resin and purified using a similar procedure to that described in Example 2.6. The peptide was
~h~r~t~ri~e~ by HPLC, mass spectroscopy and amino acid analysis in a similar manner to that
described for Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~ining 0.1%
TFA, using a 20-40% acetonitrile gradient over 20 minutes, flow rate 1.0 ml/minute) retention
time= 11.57 minutes; mass Spectrometry, m/e (ES+) 1006.4 (MH ), 503.8 (M+2H +3, 514.8
(M+H~+Na+); amino acid analysis gave: Ala 4.19, Arg 0.91, Thr 0.74.
E~caml~le 21. Phv-Ala-Arg-Ala-Arg-Thr-IIb-Azgly-Papa-NH2 (SEQ ID NO: 22)
The first part of the synthesis was performed m~nn~lly using similar methodology to that
described for Example 2.6, using HBTU/DIPEA activation and coupling and deprotecting the
Fmoc-protected amino acids and Fmoc-IIb-OH in the ~l.,pl,ate order, except for
inco~o.~Lion of the Azgly residue. The Azgly residue was incorporated by coupling with
Fmoc-Azgly-OSu (obtained as described in EP 518655) (4 equivalents) with HOBT (0.25
equivalents) in DMF at at 50~C for 4 hours. There was thus obtained Fmoc-IIb-AzGly-Papa-
NE~-resin. The peptide resin was then transferred to an automated synthesiser (ABI 431 ) and
the r~m~inin~s residues coupled following the m~nllf~cturer's recommended conditions for
single acylations inco-l-o~ g HBTU/HOBT çh~mictry. The peptide was cleaved from the
resin and purified using a similar procedure to that described in Example 2.6. The peptide was
char~rter-~ed by HPLC, mass spectroscopy and amino acid analysis in a similar manner to that
described for Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~ining 0.1%
TFA, using a 15-35% acetonitrile gradient over 20 minutes, flow rate 1.0 ml/minute) retention
time= 8.10 minlltes; mass Spectrometry, rn/e (negative electrospray (ES-)) 1076.4 (MH-);
amino acid analysis gave Ala 2.10, Arg 2.00, Thr 0.99
Example 22. Phv-Ala-Arg-Ala-Arg-Thr-IIb-Azgly-NH2 (SEQ ID NO: 23)
An analogous procedure to that described in Example 21 was used. The first part of the
synthesis was performed mz~nnzllly using HBTU/DIPEA activation, to give Fmoc-Ilb-Azgly:
NH-resin. The peptide resin was then transferred to an automated synthe~i~er (ABI 430A) and
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W O 97/31023 PCT/GB97/00438 -50-
the ~ residues coupled following the mslnllf~tllrer~s recommended conditions for
sing}e acylations incorporating HBTU/HOBT chçmi.51ry. The peptide was cleaved from the
resin and purified by preparative RP-HPLC, using a similar procedure to that described in
F.x~mrl~ 2.6. The peptide was characterised by HPLC, mass spectroscopy and arnino acid
5 analysis in a similar manner to that described for Example 1.4; RP-HPLC (eluting with
acetonitrile and water c~ l;llg 0.1% TFA, using a 20-35% acetonitrile gradient over 20
minrltes, flow rate l.0 ml/minute.) retention time = 7.26 minlltP~; mass spectrometry, rn/e
(ES+) 945.6 (MH ), 473.4 (M+2H ); amino acid analysis gave Ala 2.08, Arg 2.00, Thr 0.78.
ExamPle 23. Phv-Ala-Arg-Ala-IIb-Ala-Arg-Ala-Papa-NH2 (SEQ ID NO: 24)
An analogous procedure to that described in Example 2.6 was used, except that the whole of
the peptide assembly was performed manually using HBTU/DIPEA activation, coupling
Fmoc-Papa-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-IIb-OH and 5-phenylvaleric acid
in the ~l..o,~,liate order. The peptide was cleaved from the resin using a similar procedure to
15 that described in Exarnple 2.6. The product was purified using preparative RP-HPLC in a
similar manner to that described for Exarnple 1.4, eluting with a gradient of acetonitrile-water
cont~inin~ 0.1%TFA (15-30% acetonitrile) over 90 min~ltf~s at a flow rate of 10ml/minute.
The product was characterised by HPLC, mass spectroscopy and amino acid analysis in a
similar marmer to that described for Example l .4; RP-HPLC (eluting with acetonitrile and
water cont:~inin~ 0.1% TFA, using a 15-30% acetonitrile gradient over 30 minl~t~s7 fiow rate
1.0 ml/minute) retention time = 20.02 min-lt.o~; mass spectrometry, rn/e (ES+) 1061.6 (MH ),
531.4 (M+2H++3; arnino acid analysis gave Ala 4.16, Arg 2.00, IIb present.
Exampie 24.
3-(2-Cyanobenzo[blthiophen-5-yl)propionyl-Ala-Arg-Ala-IIb-Ala-Arg-Ala-Papa-NH2
(SEQ ID NO: 25)
The synthesis and purification was p~lrol,lled m~nuz3l1y using similar methodology to that
described for Example 1.4, coupling and deprotecting the Fmoc-Papa-OH (used instead of
Fmoc-Pip-OH), Fmoc-protected arnino acids and Fmoc-IIb-OH in the ~p~ pl.ate order, and
using 3-(2-cyanobenzo[b~thiophen-5-yl)propionic acid instead of 5-phenylvaleric acid. The
product was purified using pl~aldtive RP-HPLC in a similar manner t-o that described for
Example 1.4, eluting with a gradient of acetonitrile-water conl~ining 0.1%TFA (l5-40%
CA 02242809 1998-07-09
W O 97131023 PCT/GB97100438
~c~etonifrile) over 60 .. i~ s at a flow rate of lOml/minute. The product was (~h~r~ct~on~e-l by
HPLC, mass spectroscopy and amino acid analysis in a sirnilar manner to that described for
Example 1.4; RP-HPLC (eluting with acetonitrile and water co..l~ g 0.1% TFA, using a
10-50% ~et. nitrile gradient over 30 minllte~, flow rate l.Oml/minute) retention time = 17.58
min~lt~s, mass spectrometry, m/e (ES+) 1114.7 (MH+); amino acid analysis gave Arg 2.86,
Ala 4.12, lIb 1.00.
3-(2-Cyanobenzo[b]thiophen-5-yl)propionic acid was obtained as follows:
(a) Raney Nickel (lg) and hydrazine hydrate (3ml) was added to ethyl 5-
nikobenzo~b]thiophene-2-carboxylate (25g) (obtained as described in Syn. Comrn., (1991),
21, 959-964) in ethanol (500ml) at 60~C. A vigorous reaction ensued and the mixture
refluxed. Further portions of Raney Nickel (1 g) and hydrazine hydrate (3ml) were added at
10 minute intervals until a total of lSml had been added and the mixture was then refluxed for
90 minllt~s. The hot mixture was filtered and the filtrate evaporated. The residue was
dissolved in hot ethanol (300 ml) and activated charcoal was added. The mixture was filtered
and the filtrate evaporated to dryness to give ethyl 5-arninobenzo[b]thiophene-2-carboxylate
(18g) as a pale yellow solid. The solid was added to a stirred ~ cLule of concentrated
hydrochloric acid (50ml) and water (130ml) at ~ 0~C. A solution of sodium nitrite (5.6g) in
water (20 ml) was added slowly and then the mixture was allowed to stir at 0~C for a further
30 minllt~s A solution of potassium iodide (130g) in water (200ml) was then added. The
rnixture was allowed to warm to ambient tt;,.~pel~Lture and then heated at 50~C for 30 minllt~s
The mixture was allowed to cool, extracted with chloroform (lSOml) and the organic layer
washed with sodium metabisulphite solution. The organic phase was dried (MgSO4) and
evaporated to give a brown oil. The oil was purified by chromatography on silica using a
gradient of hexane increasing to 10% ethyl acetate /hexane. The a~ ,;ate fractions were
combined and evaporated to give ethyl 5-iodobenzo~b]thiophene-2-carboxylate (14g) as an
oil; NMR (d6-DMSO): 1.35 (t, 3H), 4.4 (q, 2H), 7.8 (dd, lH), 7.9 (d, lH), 8.15 (s, lH), 8.45
(d, IH).
(b) Sodium hydroxide (Sg) in water (ISOml~ was added to ethyl 5-iodobenzo[b]thiophene-
2-carboxylate (14g) in ethanol (200ml) and the mixture was stirred for 16 hours. The mixture
was concentrated to about 150 ml by evaporation and the mixture was adjusted to ~ pH 3 by
CA 02242809 1998-07-09
W 097131023 PCT/GB97100438
-52-
addition of dilute hydrochloric acid. The Illi;l~l,UlG was then e~.a(;~id with diethyl ether (2 x
1 OOml). The combined organic extracts were dried (MgS04) and evaporated to give 5-
iodobenzo[b~thiophene-2-carboxylic acid (11.7g) as a solid; NMR (d6-DMSO): 7.8 (dd, IH),
7.9 (d, lH), 8.05 (s, IH), 8.45 (d, lH).
5 (c) Oxalyl chloride (2.7ml) and DMF (l drop) was added to 5-iodobenzo[b~thiophene-2-
carboxylic acid (3.05g) in dichloromethane (30ml), whe~ oll evolution of gas began. After
gas evolution had ceased, the mixture was evaporated to dryness and the residue was
dissolved in THF (lOml). The solution was added to con~entr~te(l aqueous arnmonia (25ml)
in portions. After the addition was complete, the mixture was stirred for 1 hour. The solid
l O which precipitated was collected by filtration, washed with water and dried under vacuum.
The solid (3g) was dissolved in DMF ~ l 5ml) and the mixture was added at 0~C to a mixture
of DMF( 20ml) and phosphoryl chloride (3g). The mixture was stirred for 2 hours. The
mixture was then added to water (60ml) and the solid which ~ d was collected by
filtration, washed with water and dried under vacuum to give 2-cyano-5-
iodobenzo[b]thiophene (2.65g) as a pale yellow powder; NMR (d6-DMSO): 7.9 (dd, lH), 8.0
{d. lH), 8.3 (s, IH), 8.45 (d, lH).
(d) Methyl acrylate ( 2g), triethylamine (2.35g) and palladium acetate (O.OSg) was added
to 2-cyano-5-iodobenzo~b]thiophene (4.8g) in DMF (25ml) and the mixture was stirred under
argon at 90~C for 30 minutes. The mixture was allowed to cool and then added to water
(50ml). The solid which precipitated was collected by filtration and dissolved in chloroforrn.
Activated charcoal was added and the mixture filtered and the filtrate dried (MgSO4). Solvent
~as removed by evaporation to give methyl 3-(2-cyanobenzo~b]thiophen-5-yl)acrylate (2.5g);
NMR (d6-DMSO): 3.75 (s, 3H), 6.7 (d, lH), 7.8 (d, lH), 8.0 (dd, lH), 8.2 (d, lH),
8.3 (d, lH), 8.85 (s, lH).
(e) Methyl 3-(2-cyanobenzo[b~thiophen-5-yl)acrylate (2.5g) was hydrogenated in THF
(20ml) using 10% palladium on carbon (O.Sg) as catalyst until the majority ofthe starting
m~t~ri~l had dis~ppehL~d by thin layer chromatography (silica plates eluting with ethyl
acetate/hexane 1:4). The mixture was filtered and evaporated to give methyl 2-
cyanobenzo[b]thiophene-~-propionate (1 .45g) co~ ..l;n~t~r~ with ~ 20% of starting material.
Sodium hydroxide (0.23g) was added to a mixture of this product ( 1 .4g) in THF ( 1 Oml) and
u ater ~3ml) and the mixture was stirred for 2 hours. The mixture was adjusted to ~ pH 5 with
concentrated hydrochloric acid and concentrated by evaporation to remove the THF present.
CA 02242809 1998-07-09
W O 97131023 PCT/GB97100438
-53-
The pH of the ~ Lule was then adjusted to ~ pH 3 with concentrated hydrochloric acid and
the mixture ~.~tr~cte~l with dichloromPth~n~ The extract was dried (MgSO4) and evaporated.
T~e residue was purified by reverse phase c~ dlography using Cl8 support and a gradient
of 40% methanol /water increasing to 75% m~th~nolJwater. The a~lol-l;ate fractions were
combined and evaporated to give 2-cyanobenzo[b]thiophene-5-propionic acid (0.28g) as a
white solid.; NMR (CDCl3): 2.88 (t, 2H), 3.1 (t, 2H), 7.4 (d, lH), 7.75 (s, lH), 7.8 (d, lH),
7.85 (s, lH).
E~cam~le 25. Phv-Arg-Ala-Ala-IIb-Ala-Arg-Ala-Papa-NH2 (SEQ ID NO: 26)
10 The synthesis was performed m~ml~lly, and the purification carried out. using similar
methodology to that described for Example 1.4, coupling and deprotecting the Fmoc-Papa-
OH (used instead of Fmoc-Pip-OH), Fmoc-protected amino acids and Fmoc-IIb-OH in the
d~pl~,pliate order. The product was characterised by HPLC, mass spectroscopy and amino
acid analysis in a similar manner to that described for Example 1.4; RP-HPLC (eluting with
15 acetonitrile and water co~ g 0.1% TFA, using a 10-50% acetonitrile gradient over 30
mim~t~s, flow rate 1.0mLfminute) retention time = 17.45 minlltes; mass spectrometry, m/e
(ES+~ 1061.9 (MH+); amino acid analysis gave Arg 1.94, Ala 4.û4, IIb 1.02.
Example 26. Phv-Arg-Ile-Ala-IIb-Ala-Arg-Ala-Papa-NH2 (SEQ ID NO: 27)
20 The synthesis was performed automatically on an automatic synthesiser (ABI 431 ) using
similar methodology to that described in Example 2.6, coupling the Fmoc-Papa-OH and
Fmoc-y~ e~;led arnino acids in the ap~ e order, with the exception that the Fmoc-Ilb-
OH was inco.~oldled m~lu~lly. The product was characterised by HPLC, mass spectroscopy
and arnino acid analysis in a similar manner to that described for Example 1.4; RP-HPLC
25 ~eluting with acetonitrile and water c~ t~ lg 0.1% TFA, using a 10-50% acetonitrile
g~adient over 30 ~ rc, flow rate 1.0ml/minute) retention time = 20.7 min~t~ ~; mass
spectrometry, m/e (ES ) 1103.8 (MH ); amino acid analysis gave Arg 1.92, Ala 3.03, IIb 0.96,
Ile 1.08.
F,Y~ pl~ 27. Phv-lle-Arg-Ala-IIb-Leu-Arg-Ala-Papa-NH2 (SEQ ID NO: 28)
The synthesis was p~ ed m~nll~lly, and the purification carried out~ using similar
methodology to that described for Example 1.4, coupling and deprotecting the Fmoc-Papa-
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-~4-
OH (used instead of Fmoc-Pip-OH), Fmoc-protected amino acids and Fmoc-IIb-OH in the
a~lup~ order. The product was ch:~r~rt~rised by ~PLC, mass spectroscopy and amino
acid analysis in a similar manner to that described for Example 1.4; RP-HPLC (eluting with
acetonitrile and water cont~in;n~ 0.1% TF~, using a 10-50% acetonitrile gradient over 30
S mimltee, flow rate 1.0ml/minute) retention time = 23.8 mimlt~c, mass spectrometry, m/e (ES~)
1145.7 (MH~); amino acid analysis gave Arg 1.98, Ala 1.92, Leu 0.99, IIe 1.09, IIb present.
E;xample 28. Phv-Ala-Arg-Ala-IIc-Ala-Ala-Ala-Papa-NH2 (SEQ ID NO: 29)
The synthesis was ~.elro~ ed m~mls~lly, using similar methodology to that described for
10 ~Fx~mrle I .4, coupling and deprotecting the Fmoc-Papa-OH (used instead of Fmoc-Pip-OH),
Fmoc-protected an~ino acids and Fmoc-IIc-OH in the ap~ol,l;ate order. The product did not
require purification by preparative HPLC. The product was characterised by HPLC, mass
spectroscopy and amino acid analysis in a similar marmer to that described for Exarnple 1.4;
RP-HPLC (eluting with acetonitrile and water co~ 0.1 % TFA, using a 20-40%
15 acetonitrile ~r~Aient over 20 minutes, flow rate 1.0ml/minute) retention time = 10.45 min~lt~s
mass ,~e~ u~.~etry, m/e (ES+) 962.5 (MH+), 492.8 (M+H+Na) ; amino acid analysis gave
Ala 4.90, Arg 1.00, IIc present.
Fmoc-IIc-OH was obtained using an analogous procedure to that described in Example
20 I fûr the ~ ion of Fmoc-IIb-OH but using glycine methyl ester hydrochloride in place of
L-alanine methyl ester hydrochloride in step I .1. The following intermediates were obtained:
Boc-(D)-Met-Gly-OMe; in 80% yield; NMR (CDC13): 1.4d (s, 9H), 2.0d (m, lH), 2.1d (m,
lEI), 2.1d (s, 3H), 2.6d (t, 2H), 3.8d (s, 3H), 4.05d (m, 2H), 4.4d (m, lH), 5.3d (bs, lH),
6.8d (bs,lH);
25 Methyl 2-[(3;O-3-OE~-[~-butyloxycarbonyl]amino)-2-oxo-pyrrolidin-1-yl]~t~et:~te; in 50%
yield; NMR (CDC13): 1.45d (s, 9H), 2.0d (m, IH), 2.6d (m, IH), 3.4d (rn, 2H), 3.8d (s, 3H),
4.05d (~, 2H), 4.1d (m, IH), 5.55 (bs, IH),
2-[(3~)-3-(~-~9-fluorenylmethyloxycarbonyl]amino)-2-oxo-pyrrolidin-1-yl]acetic acid; in
75% yield; NMR (d6-DMSO): 1.9d (m, I H), 2.3d (m, 1 H), 3.3d (m, 2H), 4.0d (q, 2H),
30 4.3d (m, 4H), 7.4d (m, 4H). 7.6d (m. 2H), 7.9d (d, 2H).
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-55-
Exam~le 29 Phv-Arg-Ala-Ala-IIb-Ala-Ala-Ala-papa-NH2 (SEQ ID NO: 30)
The synthesis was carried out autom~ticsllly on an ACT 357 automated peptide synth~ er
(using Rinlc Amide MBHA resin), coupling the Fmoc-Papa-OH and ~moc-protected amino
acids in the ~ iate order, with the exception that the the Fmoc-IIb-OH was incorporated
m~n~ lly. The automatic couplings were ~lrolllled, following the m~nllf~ct~lrer's
~econl-llended conditions for single acylations, by activation of the carboxylic acid ( I mmol)
with diisopropylcarbodiimide (1 equivalent) and HOBT (1 equivalent) in DMF for
approximately 11 minutes before transfer to the resin. The acylation was carried out for
hllately 60 minnt~ and the washing and deprotection procedures carried out in a
10 similar manner to that described in Example 2.6. The manual coupling of the Fmoc-IIb-OH
was carried out in a Bond Elut tube using HBTU/HOBT chemistry. The S-phenylvaleric acid
required a double couple to obtain a positive result by the Kaiser test. The peptide was then
cleaved from the resin and purified by plG~ald~ive RP-HPLC using similar methodology to
that described in Example 1.4. The product was characterised by HPLC? mass spectroscopy
15 and amino acid analysis in a similar matter to that described for Example 1.4; RP-HPLC
(eluting with acetonitrile and water cont~inin~ 0.1% TFA, using a 20-35% acetonitrile
gratlient over 15 minlltec, flow rate 1.0 ml/ minute3 retention time = 10.33 minl~te~; mass
spectroscopy, m/e (ES+) 976.5 (MH ), 499.9 (M+H+Na)++; amino acid analysis gave Ala
5.02, Arg 1.00, IIb 1.22.
Example30 Phv-Lys(=C(NMe2)2)-Ala-Ala-Ala-Thr-lIb-Ala-Papa-NH2 (SEQIDNO: 31)
Using an analogous procedure to that described in Example 1 5(a), but using Fmoc-Lys(Boc)-
OH in place of Fmoc-Dap(Boc)-OH, there was thus obtained, after the peptide was cleaved
f~om the resin and purification by preparative RP-HPLC, Phv-Lys-Ala-Ala-Ala-Thr-IIb-Ala-
Papa-NH2 (270mg). The peptide (270mg) was then stirred in a mixture of HBTU (94mg),
diisopropylethylamine (88 microlitres) and DMF (SOml) for 16 hours. The mixture was
e~/~o~ ed and the residue purified by ~ dldLiveRP-HPLC using a analogous procedure to
that described in Example 1 5(a). The peptide was characterised by HPLC, mass spectroscopy
and amino acid analysis in a similar manner to that described in Example 1.4; RP-HPLC(1O-
40% acetonitrile gradient over 40 minutes? flow rate 1.2ml/minute) retention time = 29.83
minllteS, mass spectroscopy, m/e (ES ) 1077.27 (MH+)~ amino acid analysis gave Thr 1.0, Ala
4.3~ Arg 1.00. IIb present; tetramethylhomoarginine present.
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-~6-
l~amples 31-33
Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NHCH2CH2-Morpholine (hY~mple 31)
(SEQ ID NO: 32)
S Phv-Arg-Ala-Ala-Ala-~hr-IIb-Ala-Papa-OCH3 (Example 32) (SEQ ID NO: 33)
Phv-Arg-Ala-Ala-Ala-Thr-~Ib-Ala-Papa-OH ~Example 33) (SE~ ID NO: 34)
H~dlv~y-l,ethyl polystyrene resin (1.67g, lmmol) was stirred at ambient temperature with
~moc-Papa-OH (l.Sg, 4rnmol), diisopropylcarbodiimide (0.628ml, 4mmol) and
dimethylaminopyridine (61mg, O.Smmol) in DMF (Sml) and dichloromethane (20ml) for 2
10 hours. The resin was filtered and washed with DMF (Sx20ml) and meth~n~l (Sx20ml) and
dried in vacuo at 40~ (yield 2.105g). The peptide resin was then transferred to an automated
synthesiser (ABI 430A) and the rem~in;ng Fmoc-protected arnino acids coupled in a manner
similar to that described in Example 2.~, to give the protected peptide resin (2.992g). The
peptide resin was then suspended in a mixture of DMF (1 Oml) and methanol (1 Oml) and 4-(2-
15 aminoethyl)morpholine (0.88ml), was added followed by a catalytic amount of potassiumcyanide (SOmg). After 7 days at ambient temperature, the mixture was filtered and the resin
was washed with DMF (Sx20ml). The resin was immediately re-suspended in a mixture of
DMF (lOml), methanol (lOml) and potassium cyanide (SOmg). Afte} 4 days, the mixture was
filtered and the resin was washed with DMF (Sx20ml). All of the filtrates and washings from
20 both tre~tn ~nf~ were combined and evaporated to dryness. After deprotection with 90%
TFA~EI2O and removal of volatile material by evaporation, the residue was purified bv
~.e~a aLive RP-HPLC, using a similar procedure to that described in Example 1.4. to give 3
products. The products were characterised as follows:
25 Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-NHCH2CHz-Morpholine (SEQ ID NO: 32)
(40mg);
RP-HPLC (10-50% acetonitrile gradient over 40 min, flow rate 1.2ml/minute) retention time
= 19.~ min~-t~ ~ mass spectrometry, m/e (ES+) 1119.8 (MH)~, amino acid analysis gave Thr
~.58, Ala3.84, Arg 1.16, Ilb 1.00;
~hv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-OMe (SEQ ID NO: 33) (37mg);
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RP-HPLC (10-50% acetonitrile gradient over 40 min, flow rate 1.2ml/minute) retention time
= 27.7 minute~s; mass :~e~ etry, m/e ~ES+) 1021.6 (MH)+; amino acid analysis gave Thr
0.61, Ala 3.80, Arg 1.22, IIb 0.97;
5 Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-OH (SEQ ID NO: 34) (155mg);
RP-HPLC (10-50% acetonitrile gradient over 40 min, flow rate 1.2ml/minute) retention time
= 24.2 minllte~; mass spectrometry, m/e (ES+) 1007.5 (MH); amino acid analysis gave Thr
0.62, Ala 3.78, Arg 1.20, IIb 0.98.
10 ExamPle 34.
Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-N(CH2CH2)2N-(CH2)20(CH2)20H
(SEQ ID NO: 35)
4-[2-(2-hydroxyethoxy)ethyl]~ip~ ine (235 mg) was added to a mixture of Phv-Arg-Ala-
Ala-Ala-Thr-IIb-Ala-Papa-OH (135mg), HBTU (51 mg), DIPEA (47 microlitres) and DMF
15 (10 ml) and the ~llixlule was stirred for 60 minllte~ Volatile material was removed by
evaporation, and the residue was purified by ~ ~ali~e HPLC, using a similar procedure to
the described in Example 1.4, to give Phv-Arg-Ala-Ala-Ala-Thr-IIb-Ala-Papa-
N(CH2CH2)2NCH2CH2- OCH2CH2OH (SEQ ID NO: 34) (115mg). The peptide was
characterised by HPLC, mass spectrometry and amino acid analysis in a similar manner to
20 that described for Example 1.4; RP-HPLC (eluting with acetonitrile and water cont~ining
0.1%TFA, using a 10-50% acetonitrile gradient over 40 minntes, flow rate 1.2ml/minute)
retention time = 18.83 ...il~ , mass ~e~ letry, m/e (ES+) 1163.8 ~MH); arnino acid
analysis gave Thr 0.62, Ala 3.80, Arg 1.22, IIb 0.97.
25 Example 35
The compounds of the invention may be ~llrnini~t.qred for therapeutic or prophylactic use to
warm-blooded animals such as man in the form of conventional pharm~.~eT-tical compositions,
a typical example of which includes the following:-
30 Iniectable Solution0.01 to 100 mg of active ingredient is dissolved in up to 2 ml of ari aqueous injection vehicle
to give a concentration of active ingredient between 0.01 to 100 mg/ml. The aqueous
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WO 97/31023 PCT/GB97/00438
-~8-
injection vehicle is buffered to a pH bclweell S and 8 using a l~h~rrn~.eufically acceptable
buffer (for example, rho,crh~t~, or acetate) and contains a rh~rrn~re~lfically acceptable
tonicity adjnetnn~nt agent (for exarnple, sodiurn chloride or dextrose3 added to achieve
isotonicity. The vehicle may optionally also contain other rh~rrn~eutically acceptable
excipients such as solubilising agents (for example, DMSO, ethanol, propylene glycol or
polyethylene glycol) preservatives and antioxi(l~nt.c The active ingredient may typically be
an ~xample described hereinbefore and may conveniently be present as a pharrnaceutically
acceptable salt.
HS70147WO
JJHIIFEB97
,
CA 02242809 1998-07-09
W 097/31023 PCTIGB97/00438
_ ~;9 _
Chemical Formulae
P Rt_R2_R3--R4
A O ~
H ~ s\ N~ \
O Ra O Ra
Il lla
H ~ \
llb llc
- ~\N ~¦~ ~N ~!Ç\N ~A
~ Rb I ~ Rb
111 Illa
~ CH3
Illb
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_60-
~u~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ZENECA LIMITED
(B) STREET: 15 STANHOPE GATE
(C) CITY: LONDON
(E) COUNTRY: UNITED KlN~ M
(F) POSTAL CODE (ZIP): WlY 6LN
(G) TELEPHONE: Q171 304 5000
(H) TELEFAX: 0171 304 5151
(I) TELEX: 0171 834 2042
(ii) TITLE OF INVENTION: PEPTIDE DERIVATIVES
(iii) NUMBER OF SEQUENCES: 36
~iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) C~..~U1~: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPC
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9603855.9
(B) FILING DATE: 23-FEB-1996
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9620819.4
(B) FILING DATE: 05-OCT-1996
(2) INFORMATION FOR SEQ ID NO: 1:
~QU~N~ CHARACTERISTICS.
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~FnNF~C: 9 in~le
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylp~nr~n~yl-Ala''
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propar.~yl]-Ala-
piperidine-4-carbaxamide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
Xaa Ala Ala Lys Val Xaa
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W O 97/31023 PCT/GB97/00438
-61-
l 5
(2~ INFORMATION FOR SEQ ID NO: 2:
( i ) ~UU~N~ CH~RACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) sTp~Nn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/~Y: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/notez ''5-Phenylpentanoyl-Ala
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) 10CATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(5)-2-(6-oxo-1,7-diazaspiro[4.4]non-7-yl)propanoyl]-Ala-
piperidine-4-carboxamide"
(Xi) ~E~U~N~ DESCRIPTION: SEQ ID NO: 2:
Xaa Ala Ala Lys Val Xaa
l 5
(2) INFORMATION FOR SEQ ID NO: 3:
( i ) ~uu~N~E CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTRANn~nN~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
lix) FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:4
~D) OTHER INFORMATION:/product= "OTHER"
/note= "[~S)-2-(~R)-3-amino-2-oxopyrrolidin-l-yl~propanoyl]-Ala"
~ix~ FEATURE:
~A~ NAME/KEY: Peptide
(B~ LOCATION:6
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Ala-piperidine-4-carboxamide"
(Xi) ~UU~'N~ DESCRIPTION: SEQ ID NO: 3:
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-62-
Xaa Lys Ala Xaa Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 4:
i ) ~U~N~ CHARACTERISTICS:
tA) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) ST~AN~ )N~-~.s: single
(D) TOPOLOGY: linear
( ii ) MOT.~CUr.F TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ile"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
n [ (S) -2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
:lmi rlnph~nylacetamide ~
(Xi) ~:y~:N~ DESCRIPTION: SEQ ID NO: 4:
Xaa Ala Ala Arg Thr Xaa
1 5
~2) INFORMATION FOR SEQ ID NO: 5:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) STRANl~ JN~:~S: single
tD) TOPOLOGY: linear
~ii) M~T~T'~uT~T~ TYPE: peptide
~ix) FEATURE:
~A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION:~
~D) OTHER INFORMATION:/product= "OTHER"
/note=
~l[(s)-2-(~R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl~-Ala
piperidine-4 -~rhn~mt de"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Xaa Ala Ala Ala Val Xaa
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WO 97131023 PCTIGB97100438
-63-
~2) INFORMATION FOR SEQ ID NO: 6:
(i) ~UUb:N~: CHARACTERISTICS:
(A3 LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~ N~:~S: single
(D) TOPOLOGY: linear
(ii) MOLECU~E TYPE: peptide
(ix) FEAlUKE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/3CEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S3-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
aminophenylacetamide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 7:
:Uu~N~ CHARACTERISTICS:
(A) LENGT3~: 6 amino acids
(B) TYPE: amino acid
(C) ST~ANI~ N~:~c: slngle
(D) TOPOLOGY: linear
(ii3 MOLECULE TYPE: peptide
(ix~ FEATu~E:
(A) N~ME/~EY: Peptide
(B) LOCATION:1
(D3 OTHER INFORMATION:/product= "OTHER~
/note- "5-Phenylpentanoyl-Ala"
(ix) FEATURE:
(A) NAME/ ~Y: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
I~(S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl)-Ala-4-
aminophenylacetamide~"
(xi) ~hUu~:N~: DESCRIPTION: SEQ ID WO: 7:
Xaa Ala Ala Arg Val Xaa
1 5
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-64-
(2) INFORMATION FOR SEQ ID NO: 8:
CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) sTR~Nn~nN~ 3 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[~S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Gly-4-
aminophenylacetamide]"
(xi) ~Q~N~ DESCRIPTION: SEQ ID NO: 8:
Xaa Arg Ala Arg Val Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOC~TION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-
piperidine-4-r~rhr~ide]"
(xi) SEuu~N~ DESCRIPTION: SEQ ID NO: 9:
Xaa Ile Ala Arg Val Xaa
(2) INFORMATION FOR SEQ ID NO: lQ
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W O 97/31023 PCT/GB97/00438
~i) ~hUUhN~h CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~n~.~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
~A) NAME/KEY: Peptide
~B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
(ix) FEATURE:
~A) NAME/KEY: Peptide
~B) LOCATION:6
~D) OTHER INFORMATION:/product= "OTHER"
/note=
"[~S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Ala-4-
aminophenylacetamide]"
(xi) ~hUUhN~h DESCRIPTION: SEQ ID NO: 10:
Xaa Arg Ala His Val Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 11:
hUUhN~h CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRPN~ )N~:~S: single
~D~ TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
(ix) FEATURE:
~A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
~ix) FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
''I(S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Ala-
piperidine-4-carboxamide]"
(Xi) ~h~UhN-~h DESCRIPTION: SEQ ID NO: 11:
Xaa Ala Asn Arg Val Xaa
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 6 amino acids
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(B~ TYPE: amino acid
(C) STRp~Nr~nN~-cs: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylp~n~n~yl-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "~(S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= nAla-piperidine-4-~h~lde"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Xaa Arg Ala Xaa Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 13:
(i) ~u~: CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) STR.~NI 11-:~ )Nl CS: ~ingle
ID) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= 'l5-phenylrent~n~yl-Ala
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Gly-3-
~minopropylamine~"
(xi ) ~U~N~ DESCRIPTION: SEQ ID NO: 13:
Xaa Arg Ala Ala Thr Xaa
l 5
(2) INFORMATION FOR SEQ ID NO: 14:
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( i ) ~U~N~: C8ARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STT~z~Nl IICI~NI~ C .C single
(D) TOPOLOGY: linear
( ii ) ~QT.~TTT.T' TYPE: peptide
(ix) FEATURE:
(A) NAME~KEY: Peptide
(B) LO QTION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= ~'5-Phenylpentanoyl-Ala"
(ix) FEATURE:
(A) NA~ME/KEY: Peptide
tB) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Gly-3-
aminopropylg--~n;~;n~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Xaa Arg Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(i) ~QU~N~' CHARACTERISTICS:
(A~ LENGT8: 6 amino acid~
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B~ LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylp~nr~noyl-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Gly-NHethyl"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Xaa Arg Ala Ar~ Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 16:
( i ) ~h~U~'N~' CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
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(C) STRANn~nN~c~: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ix) FEATURE:
(A) NAME~REY: Peptide
(B) LOCATION:l
~D) OTHER INFOR~7ATION:/product- "OTHER"
/note= "5-Phenylpentanoyl-3-g-~ni~
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
aminophenylacetamide]"
(xi) ~EYU~.N~ DESCRIPTION: SEQ ID NO: 16:
Xaa Ala Ala Ala Thr Xaa
1 5
~2) INFORMATION FOR SEQ ID NO: 17:
(i) ~UU~N~ CHARACTSRISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: pep~ide
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product- "OTHER"
/note= "5-Phenylpe~tanoyl-3-(tetramethylyuanidino)Ala"
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
nmi nr1rhf~nylacetamide,7 "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 18:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGT~: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: 8 inyle
(D) TOPOLOGY: linear
,
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tii) MOr.~Ur.~ TYPE: peptide
(ix) FEATURE:
P (A) NAME/KEY: Peptide
~B) LOCATION:1
~D) OTHER INFORMATION:/product- "OTHER"
_ /note= "5-Phenylp~n~n~yl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
~D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Gly-4-(2-
aminocthyl)morrh~l; n~ "
(Xi) s~Qu~N~ DESCRIPTION: SEQ ID NO: 18:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
( i ) ~UU~:N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRA~ N~:~S: single
(D) TOPOLOGY: linear
(ii) M~L~ TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"~(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl~-Gly-piperazin-
~-yl.CH2CH20CH2CH20H "
(Xi) ~U~N~: DESCRIPTION: SEQ ID NO: 19:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 20:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acidA
(B) TYPE: amino acid
(C) STRAN~N~'~S: single
(D) TOPO~OGY: linear
(ii) MOLECULE TYPE: peptide
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~ix~ FEATURE:
(A) NAME/KEY: Peptide
~B) LOCATION:1
(D) OT8ER INFORMATION:/produCt= "OT8ER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Gly-4-
aminophenethylgl-~n;,~;nf,~
(xi) ~:Uu~N~ DESC~IPTION: SEQ ID NO: 20:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 21:
i ) 5k~U~'N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTRANnFnNEqs single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OT8ER"
/note= "5-Phenylpentanoyl-Ala'~
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
aminophenylacetamide"
(xi) ~k~U~N~: DESCRIPTION: SEQ ID NO: 21:
Xaa Ala Ala Arg Thr Xaa
1 5
(2~ INFORMATION FOR SEQ ID NO: 22:
(i) ~kQ~ ~'N~- CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(ix) FEATURE:
(A) NAME/REY: Peptide
(B~ LOCATION:1
(D) OTHER INFORMATION:/product= OTHER
/note= 5-Phenylpentanoyl-Ala
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product- OTHER
/note=
l(S)-2-((R)-3-amino-2-oxopyrro~idin-1-yl)propanoyl~-[NH-NH-CO~-4-
Am; n~-phF~nylacetamide"
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO: 22:
Xaa Arg Ala Arg Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 23:
yu~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~N~:~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B~ LOCATION:1
(D) OTHER INFORMATION:/product= OTHER
/note= 5-Pheny~ p~nt ~n~yl - Ala
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= OTHER
/note=
[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl~propanoyl~-NH-NH-CONH2
(Xi ) ~UU~N~ DESCRIPTION: SEQ ID NO: 23:
Xaa Arg Ala Arg Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 24:
( i ) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
( ii ) MOLT~rTT~T~ TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
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(D~ OTHER INFORMATION:/product= "OTHER"
/note- "5-Phenylpentanoyl-Ala~'
~ixl FEATURE:
(A~ NAME/KEY: Pep~ide
(B~ LOCATION:4
(D~ OTHER INFORMATION:/product= "OTHER"
/note= ''[(S~-2-((R~-3-amino-2-v~vpyrrvlidin-l-yl)propanoyl]-Ala~
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B~ LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Ala-4-~norh~nylacetamide"
(Xi~ U~N~ DESCRIPTION: SEQ ID NO: 24:
Xaa Arg Ala Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 25:
(i) ~Uu~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B~ TYPE: amino acid
(C~ STRANnR~NR~ single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix~ FEATURE:
(A~ NAME/KEY: Peptide
(B~ LOCATION:1
(D~ OTHER INFORMATION:/product= "OTHER"
/note- 'l3-(2-cyanobenzotblthiophen-5-yl)propanoyl-Ala~
(ix~ FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/notez "~(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl~propanoyl]-Ala"
(ix) FEAluKE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D~ OTHER INFORMATION:/product= nOTHER"
/note= "Ala-4-~min~ph~nylacetamide"
(xi~ ~Uu~N~ DESCRIPTION: SEQ ID NO: 25:
Xaa Arg Ala Xaa Arg Xaa
1 5
r2~ INFORMATION FOR SEQ ID NO: 26:
( i ~ S~UU~:N~ CHARACTERISTICS:
(A~ LENGTH: 6 amino acids
(B~ TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: peptide
~ix) FEATURE:
(A~ NAME/KEY: Peptide
r (8) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= nOTHER"
/note= "[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product- "OTHER"
/note= ~Ala-4-~min~rh~nylacetamide~
(xi) ~Uu~N~: DESCRIPTION: SEQ ID NO: 26:
Xaa Ala Ala Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 27:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B~ TYPE: amino acid
(C) sTRANn~nN~cs single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER~
/note= ''5-Phenylpentanoyl-Arg
(ix~ FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "[(S)-2-(~R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Ala-4~ norh~nylacetamide"
(xi) ~UUkN~ DESCRIPTION: SEQ ID NO: 27:
Xaa Ile Ala Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 28:
_
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(i) ~QU~N-~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPB: amino acid
(C) STR~N~ )N~:~cc: single
(D) TOPOLOGY: linear
(ii) ~T.T'~TT,R TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ile"
(ix) FEATURE:
tA) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Leu"
(ix) FEATURE:
(A) NAME/REY: Peptide
( B ) LOCATION:6
(D) OTHER INFORMATION:/product= "OTH~R"
/note= "Ala-4-~min~ph~nylacetamide"
(xi) ~iEyu~N~ DESCRIPTION: SEQ ID NO: 28:
Xaa Arg Ala Xaa Arg Xaa
~2) INFORMATION FOR SEQ ID NO: 29:
( i ) ~ hyu ~:NC~ CHARACTERISTICS:
(A) LENGTH: 6 amino acid3
(B) TYPE: amino acid
(C) sTR~NnRn~Rss single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "[((R~-3-amino-2-oxopyrrolidin-1-yl~acetyl]-Ala"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= 'lAla-4-aminophenylacetamide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
.;
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Xaa Arg Ala Xaa Ala Xaa
l 5
(2) lN~ TION FOR SEQ ID NO: 30:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
~B) TYPE: amino acid
(C) STRANv~N~S: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
tA) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylp~n~Annyl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl~-Ala~
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Ala-4-Aminnph~nylacetamide"
(xi) ~UU~N~: DESCRIPTION: SEQ ID NO: 30:
Xaa Ala Ala Xaa Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 31:
(i) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTRANnEnN~cs: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-tetramethylhomoArg-"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
- /note-
ll[(s)-2-~(R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl~-Ala-4
aminophenylacetamide"
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~Xi) ~UU~:N~' DESCRIPTION: SEQ ID NO: 31:
Xaa Ala Ala Ala Thr Xaa
1 5
~2~ INFORMATION FOR SEQ ID NO: 32:
(i) ~YU~N~' CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRAN~ N~ : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Peptide
(B) LOCATION:1
(D) OT~ER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg~'
~ix) FEATURE:
tA) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-~4-
~in~ph~nyl C~2 CO NH.CH2~CH2.morpholine"
(xi) ~Qu~ DESCRIPTION: SEQ ID NO: 32:
Xaa Ala Ala Ala Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 33:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR ~Nn~nN~.~ ~: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEAluKE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= 'l5-phenylr~nt~n~yl-Arg
(ix) FEATURE:
(A) NAME~KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
[(s)-2-((R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Ala-4
in~ph~nylacetic acid methyl ester"
(Xi) ~uu~-N-~ DESCRIPTION: SEQ ID NO: 33:
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Xaa Ala Ala Ala Thr Xaa
1 5
(2~ INFORMATION FOR SEQ ID NO: 34:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) sTR~n~n~-~c 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg~
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
'~[(s)-2-~(R)-3-amino-2-oxopyrrolidin-l-yl)propanoyl]-Ala-4
aminophenyl acetic acid"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Xaa Ala Ala Ala Thr Xaa
1 5
~Z) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) STRA~ S: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
(D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Arg"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
~ (S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
aminophenylacetyl-piperazin-4-yl-CH2CH20CH2CH20H"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Xaa Ala Ala Ala Thr Xaa
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1 5
- (2) INFORMATION FOR SEQ ID NO: 36:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STR~Rr~ : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:l
~D) OTHER INFORMATION:/product= "OTHER"
/note= "5-Phenylpentanoyl-Dprl'
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note=
"[(S)-2-((R)-3-amino-2-oxopyrrolidin-1-yl)propanoyl]-Ala-4-
~m; nnrh~nylacetamide"
(Xi) 5~:UU~N~ DESCRIPTION: SEQ ID NO: 36:
Xaa Ala Ala Ala Thr Xaa
